Labeling and detection of post translationally modified proteins

ABSTRACT

Provided in certain embodiments are new methods for forming azido modified biomolecule conjugates of reporter molecules, carrier molecules or solid support. In other embodiments are provided methods for enzymatically labeling a biomolecules with an azide group.

CROSS REFERENCE TO RELATED APPLICATION(S)

This application is a continuation of U.S. application Ser. No.11/674,140, filed Feb. 12, 2007, and a continuation of U.S. applicationSer. No. 11/674,136, filed Feb. 12, 2007, both of which claims priorityto U.S. Provisional Application No. 60/772,221, filed Feb. 10, 2006 andU.S. Provisional Application No. 60/804,640, filed Jun. 13, 2006, thecontents of which are herein incorporated by reference in theirentirety.

FIELD OF THE INVENTION

The invention generally relates to methods of labeling posttranslationally modified biomolecules using metabolic, enzymatic, orchemical incorporation of azide or alkyne-labeled macromoleculesfollowed by chemical conjugation with paired azide, alkyne, activatedalkyne, or triarylphosphine reporter molecules appended to proteins posttranslation.

BACKGROUND INFORMATION

Protein glycosylation is one of the most abundant post-translationalmodifications and plays a fundamental role in the control of biologicalsystems. For example, carbohydrate modifications are important forhost-pathogen interactions, inflammation, development, and malignancy(Varki, A. Glycobiology 1993, 3, 97-130; Lasky, L. A. Annu. Rev.Biochem. 1995, 64, 113-139. (c) Capila, I.; Linhardt, R. J. Angew.Chem., Int. Ed. 2002, 41, 391-412; Rudd, P. M.; Elliott, T.; Cresswell,P.; Wilson, I. A.; Dwek, R. A. Science 2001, 291, 2370-2376.) One suchcovalent modification is O-GlcNAc glycosylation, which is the covalentmodification of serine and threonine residues by D-N-acetylglucosamine(Wells, L.; Vosseller, K.; Hart, G. W. Science 2001, 291, 2376-2378;Zachara, N. E.; Hart, G. W. Chem. Rev. 2002, 102, 431). The O-GlcNAcmodification is found in all higher eukaryotic organisms from C. elegansto man and has been shown to be ubiquitous, inducible and highlydynamic, suggesting a regulatory role analogous to phosphorylation.However, the regulatory nature of the modification (i.e., dynamic, lowcellular abundance) also represents a central challenge in its detectionand study.

A common method to observe O-GlcNAc involves labeling proteins withβ-1,4-galactosyltransferase (GalT), an enzyme that catalyzes thetransfer of [³H]-Gal from UDP-[³H]galactose to terminal GlcNAc groups(Roquemore, E. P.; Chou, T. Y.; Hart, G. W. Methods Enzymol. 1994, 230,443-460). Unfortunately, this approach is expensive, involves handlingof radioactive material, and requires exposure times of days to months.Antibodies and lectins offer alternative means of detection, but theycan suffer from weak binding affinity and limited specificity (Snow, C.M.; Senior, A.; Gerace, L. J. Cell Biol. 1987, 104, 1143-1156; Corner,F. I.; Vosseller, K.; Wells, L.; Accavitti, M. A.; Hart, G. W. Anal.Biochem. 2001, 293, 169-177).

Isolated or synthesized antibodies, such as IgGs, are usedtherapeutically and for diagnostic and research purposes. By labelingantibodies with detectable labels, such as, for example, fluorophores,antibodies can be used to specifically detect target biologicalmolecules or cells. Antibodies may also be tagged with binding reagents,such as, for example, biotin, so that they may be used to specificallybind target biological molecules or cells, followed by purification ofthe biological molecule or cell by using a reagent that binds to thetagged antibody, for example, streptavidin. Antibodies have generallybeen labeled at cysteine or lysine residues, which may often be presentin the Fab, or binding portion of the antibody. Adding tags or labels inthis region may disrupt or at least alter the binding properties of theantibody. Further, it is often difficult to quantitate the number oflabeled molecules attached to each antibody.

One important class of glycoproteins is antibodies. Therapeuticmonoclonal antibodies (Mabs) have become indispensable drugs to combatcancer, rheumatoid arthritis, macular degeneration, and other diseasesor conditions. However, antibodies generated in non-human cell lines mayhave antigenic features recognized as foreign by the human immunesystem, limiting the antibodies' half-life and efficacy. Incorporatinghuman IgG sequences into transgenic mice has reduced, but noteliminated, immunogenicity problems. Besides the protein sequence, thenature of the oligosaccharides attached to the IgG has a profound effecton immune-system recognition. Because glycosylation is cell typespecific, IgGs produced in different host cells contain differentpatterns of oligosaccharides, which could affect the biologicalfunctions. Even where cells, such as human embryonic stem cells, aregrown on mouse feeder layers in the presence of animal-derived serumreplacements, the cells incorporated a nonhuman, and immunogenic, sialicacid, and the sialic acid was then found on the cell surface. (Martin,M. J., et al., Nature Medicine, 2005, 11:228-232). Although thetherapeutic antibody industry has tried to avoid these problems byproducing less antigenic IgG with defucosylated oligosaccharides,defucosylated antibodies are not equivalent to humanized antibodies, andmay still have immunogenecity issues, as well as having differenthalf-lives than natural human antibodies.

Metabolic oligosaccharide engineering refers to the introduction ofsubtle modifications into monosaccharide residues within cellularglycans. Researchers have used metabolic engineering to disrupt glycanbiosynthesis, chemically modify cell surfaces, probe metabolic fluxinside cells, and to identify specific glycoprotein subtypes from theproteome. (reviewed in Dube, D. H., and Bertozzi, C. R., Current Opinionin Chemical Biology, 2003, 7:616-625).

There is a need for antibodies that have tags or labels at sites otherthan the binding region, and for antibodies that may be easily labeledusing simple and efficient chemical reactions. There is also a need forantibodies that have post-translational modifications that are more likehuman antibodies.

SUMMARY OF THE INVENTION

Provided in certain embodiments are methods for enzymatically labeling aglycoprotein with an azide moiety. In one aspect a glycoprotein iscontacted with UDP-GalNAz in the presence of an appropriate enzyme. Inone aspect the enzyme is Gal T. In another aspect the enzyme is amodified Gal T enzyme. These azido modified glycoproteins can then beconjugated t a wide variety of reporter molecules, carrier molecules orsolid supports provided that they contain an azide reactive group. Inone aspect the azide reactive group is a terminal alkyne such that theconjugation reaction utilizes copper (I) catalyzed cycloadditionchemistry. In another aspect the azide reactive group is a phosphinesuch that the conjugation reaction performed is a Staudinger ligationtype reaction.

In another embodiment is provided a method for forming a reportermolecule-glycoprotein conjugate with an immobilized azido modifiedglycoprotein. Glycoproteins are modified with azido sugars eithermetabolically or enzymatically and then immobilized on a solid orsemi-solid matrix. In one embodiment the solid or semi-solid matrix is aslide, an array, polymeric particle or a gel matrix. In a particularaspect the azido modified glycoproteins are separated by gelelectrophoresis or capillary electrophoresis. The immobilized azidomodified glycoprotein is contacted with an azide reactive reportermolecule wherein the conjugate is formed either in a click chemistrytype reaction or a Staudinger ligation type reaction. Subsequently theglycoprotein conjugate is detected after illumination with anappropriate wavelength.

The azide-alkyne [3+2] cycloaddition is a chemoselective ligationreaction that is catalyzed by the addition of copper (I). Although thereaction has been applied to a variety of different bioconjugationreactions over the past several years, it has never been applied to thein-gel fluorescence detection of modified proteins. The procedureinvolves the selective tagging of proteins with reactive probescontaining either azide or alkyne groups. The reactive probes can betoward total proteins, such as those that label specific amino acidslike lysines or cysteines, or they can label (or derivatize)post-translationally modified amino acids within the proteins, such asphosphoamino acids or glycosylated amino acids. Additionally, proteinmodification can take place in vivo by metabolic labeling. This involvesfeeding cultured cells, bacteria, plants, or animals tagged metabolicprecursors that are incorporated into specific molecules by theintracellular enzymatic machinery. Primary protein labeling can beperformed with an azide or alkyne probe as long as the detection iscarried out with the alternate of the pair. Once the primary proteinlabeling step is completed, cellular extracts (or fractions) areseparated by 1-D or 2-D gel electrophoresis.

The methods described for detection of proteins in gels can be completedwithin 3-4 hours, or less. The method is devoid of the typical problemsencountered with antibody detection on blots including the requirementto optimize primary and secondary antibody concentrations and issues ofnon-specific binding of the antibodies. Finally, it is well-documentedin-gel digested proteins are more compatible with detection by massspectrometry than are electroblotted proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Shows metabolic labeling with unnatural azido sugars.

FIG. 2: Shows metabolic labeling and “click” detection of glycoproteinsubclasses A) schematically and B) detection after separation on a gel.

FIG. 3: Shows separation of Ac₄GlcNAz-treated soluble Jurkat cellproteins by 2-D gels and controls compared to the same gel stained withSYPRO® Ruby total protein stain.

FIG. 4: Shows in gel detection of 40 and 50 kD azide-labeled modelproteins, which were first labeled with a fluorescent alkyne tag andthen separated on the gel.

FIG. 5: Shows Labeling Efficiency of 40 and 50 kd Azide-Labeled ModelProteins is Unchanged in Complex Protein Extracts.

FIG. 6: Shows the Enzymatic Labeling and Detection of α-crystallinO-GlcNAc.

FIG. 7: Shows the Comparison of GalT1 Enzyme Labeling with a-O-GlcNAcMonoclonal Antibody CTD 110.6.

FIG. 8: Shows the Multiplex Detection of O-GlcNAc Proteins,Phosphoproteins and Total Proteins in the Same 2-D gel.

FIG. 9: Shows Multiplexed Western Blot Detection of O-GlcNAc andCofilin.

FIG. 10: Shows the Differential Detection of O-GlcNAc Modified Proteinsin Control and Inhibitor-Treated Cultured Cell Extracts.

FIG. 11: Shows the A) UDPGalNAz in conjunction with GalT as a novelmethod for labeling antibodies via reaction of the terminal OGlcNAcmolecules present on antibody carbohydrates (Scheme 2) and B) if noOGlcNAc sugars are present on an antibody, then use of the Endo-H(endo-β-N-acetylglucosaminidase H) enzyme will be used to generate atruncated chain which terminates with one N-acetylglucosamine residue.

FIG. 12: Shows structures of four present (A-D) and two potential (E,F)alkyne fluorophores that can be used to label biomolecules using themethods of the invention: A) TAMRA-alkyne; B) Alexa 488-Alkyne; C) Alexa633-Alkyne; D) Alexa 532-Alkyne, E) a potential fluorogenic alkyne; F) apotential fluorogenic alkyne.

FIG. 13: shows the results of in-gel staining using the TAMRA-alkynecompound shown in FIG. 12A. Lanes 2, 3, and 4 on the left side of thegel represent cellular extracts that were labeled with azide-modifiedsugars: lane 1 is the control, non-labeled cells. On the right, controlazide labeled proteins (ovalbumin and myoglobin) (+) or non-labeledcontrols (−) are shown at varying concentrations. The results show veryefficient and selective in-gel labeling of azido-modified proteins.

FIG. 14 depicts gels showing the results of separating proteins labeledusing the click reaction with different chelators. 2.5 μg each ofazido-ovalbumin and azido-myoglobin spiked into 80 ug of unlabeledJurkat lysate was labeled with TAMRA alkyne for 2 hrs. The reactioncontained 50 mM TRIS pH8, 25% propylene glycol, 1 mM CuSO₄, 5 mM sodiumascorbate, 20 uM TAMRA alkyne. The reactions were performed with andwithout chelator (10 mM TPEN [upper left gel], EDTA [upper right gel],bathocuproine disulfonic acid (BCS) [middle left gel] or neocuproine[middle right gel]). Control reactions were performed without CuSO₄[lower left gel] or without chelator [lower right gel]. After labeling,the samples were precipitated, resolubilized in 7 mM urea/2 mMthiourea/65 mM DTT/2% CHAPS/and approximately 30 μg was analyzed on 2-Dgels (pH 4-7 IEF strips, 4-12% BIS-TRIS gels with MOPS buffer). TheTAMRA signal was imaged at 532 nm excitation, 580 long pass emission ona Fuji FLA3000 (14A) then the gels were post-stained with Sypro® Rubytotal protein gel stain (14B).

FIG. 15 depicts gels showing the results of separating proteins labeledusing the click reaction with different chelators. The samples and clicklabeling conditions are the same as for FIG. 14, except that chelatortreatments include addition of either 5 mM TPEN, BCS or Neocuproine atthe beginning of the reaction. After labeling, the samples wereprecipitated, resolubilized in LDS buffer+5 mM TCEP and serial 2-folddilutions were performed. Dilutions were loaded onto 4-12% BIS-TRIS gelswith MOPS running buffer (250 ng each of ovalbumin and myoglobin in lane1). FIG. 15A shows that the chelators reduce the background of the imagefor the TAMRA signal without compromising sensitivity. In FIG. 15B,post-staining with Sypro® Ruby total protein gel stain shows that theband resolution is much better for the samples with chelator.

FIG. 16 The samples and click labeling conditions are the same as forFIG. 14, except that chelator treatments include addition of either 7mM, 5 mM or 2 mM BCS or neocuproine. The lanes marked with an asteriskin (A) indicate reactions in which the CuSO₄ and BCS were added to thereaction and vortexed prior adding the sodium ascorbate. In all otherreactions the CuSO₄ and sodium ascorbate were added and vortexed priorto adding the BCS. The gels show that it is imperative to add the sodiumascorbate and CuSO₄ to the reaction tube and mix prior to adding thechelator. If the chelator and CuSO₄ are added and vortexed prior toadding the sodium ascorbate, the azide-alkyne labeling does not proceed,suggesting that the chelator inhibits the reduction of Cu II to Cu I.

FIG. 17: Shows the enzymatic labeling of antibodies using cliackchemistry: A) Lane 1—Goat antibody only (GAb); Lane 2—GAb with GalT1enzyme but without UDP-GalNAz Control; Lane 3—GAb with enzyme andUDP-GalNAz. Lane 4—azide-labeled ovalbumin and myoglobin controlproteins; Lane 5—MW markers unlabeled and B) Azide-labeled goatantibodies (from above) were run as a dilution series. Nanograms ofantibodies were calculated for the heavy chains only. Gels were run,stained, and imaged as described above.

FIG. 18A: Shows β-elimination in the lower trace only of RII peptide andthe upper trace shows simultaneous β-elimination and azido-thioacetateaddition of RII peptide. β-elimination removes 98 Da from the peptideand azido-thioacetate addition adds 159 Da, therefore the expected massof the addition product is 2253 Da (2192-98+159). The 1980 Da peakcorresponds to β-eliminated RII with the N-terminal alanine cleaved. The1655 Da peak corresponds to hydrolysis of RII at the phosphoserineresidue and is not observed in the addition sample.

FIG. 18B: Shows β-elimination on the lower trace only of RII peptide andthe upper trace shows simultaneous β-elimination and azido-amineaddition of RII peptide. β-elimination removes 98 Da from the peptideand azido-amine addition adds 142 Da, therefore the expected mass of theaddition product is 2236 Da (2192−98+142). Another addition productcorresponding to +130 Da (expected mass=2224) is observed. PDS MALDIanalysis shows the same fragment masses as for the 2236 productsuggesting that the products are related. The 1980 Da peak correspondsto β-eliminated RII with the N-terminal alanine cleaved. The 1655 Dapeak corresponds to hydrolysis of RII at the phosphoserine residue andis observed in lesser amount in the addition sample than in theβ-elimination-only sample.

FIG. 19 Shows the results of fluorescent Click labeling of live andfixed cells

FIG. 20: Shows gel analysis of cell surface versus total glycoproteinsubclasses. Lanes 1 and 4: Control unfed; Lane 2: GalNAz fed, surfacelabeled live cells, no perm; Lane 3: GalNAz fed, total cell lysateslabeled; Lane 5: ManNAz fed, surface labeled live cells, no perm; Lane6: ManNAz fed, total cell lysate labeled.

FIG. 21: shows the results of gel analysis of labeled glycoproteins inlive animals. The upper panels below show 1-D gels of labeled tissueproteins from heart muscle (H), liver (L), or kidney (K). Lower panelsshow same gels after post-staining with SYPRO Ruby.

DETAILED DESCRIPTION OF THE INVENTION Definitions and Abbreviations

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to specific compositionsor process steps, as such may vary. It must be noted that, as used inthis specification and the appended claims, the singular form “a”, “an”and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a ligand” includes aplurality of ligands and reference to “an antibody” includes a pluralityof antibodies and the like.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention is related. The following terms andabbreviations (Table 1) are defined for purposes of the invention asdescribed herein.

TABLE 1 List of Abbreviations Abbreviation Term. GalNAzN-alpha-azidoacetylgalactosamine. GlcNAz N-alpha-azidoacetylglucosamine.GalNAc N-acetylgalactosamine. GlcNAc N-acetylglucosamine LOSLipooligosaccharide. ManLev N-levulinoylmannosamine. ManNAcN-acetylmannosamine. ManNAz N-alpha-azidoacetylmannosamine. ManNButN-butanoylmannosamine. ManNProp N-propanoylmannosamine. NCAM neural celladhesion molecule. PSA Polysialic acid. Endo H Endoglycosidase H Endo MEndoglycosidase M

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomers,geometric isomers and individual isomers are encompassed within thescope of the present invention.

The compounds described herein may be prepared as a single isomer (e.g.,enantiomer, cis-trans, positional, diastereomer) or as a mixture ofisomers. In a preferred embodiment, the compounds are prepared assubstantially a single isomer. Methods of preparing substantiallyisomerically pure compounds are known in the art. For example,enantiomerically enriched mixtures and pure enantiomeric compounds canbe prepared by using synthetic intermediates that are enantiomericallypure in combination with reactions that either leave the stereochemistryat a chiral center unchanged or result in its complete inversion.Alternatively, the final product or intermediates along the syntheticroute can be resolved into a single stereoisomer. Techniques forinverting or leaving unchanged a particular stereocenter, and those forresolving mixtures of stereoisomers are well known in the art and it iswell within the ability of one of skill in the art to choose andappropriate method for a particular situation. See, generally, Furnisset al. (eds.). VOGEL'S ENCYCLOPEDIA OF PRACTICAL ORGANIC CHEMISTRY5^(TH) ED., Longman Scientific and Technical Ltd., Essex, 1991, pp.809-816; and Heller, Acc. Chem. Res. 23: 128 (1990).

The compounds disclosed herein may also contain unnatural proportions ofatomic isotopes at one or more of the atoms that constitute suchcompounds. For example, the compounds may be radiolabeled withradioactive isotopes, such as for example tritium (³H), iodine-125(¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations of the compounds ofthe present invention, whether radioactive or not, are intended to beencompassed within the scope of the present invention.

Where a disclosed compound includes a conjugated ring system, resonancestabilization may permit a formal electronic charge to be distributedover the entire molecule. While a particular charge may be depicted aslocalized on a particular ring system, or a particular heteroatom, it iscommonly understood that a comparable resonance structure can be drawnin which the charge may be formally localized on an alternative portionof the compound.

Selected compounds having a formal electronic charge may be shownwithout an appropriate biologically compatible counterion. Such acounterion serves to balance the positive or negative charge present onthe compound. As used herein, a substance that is biologicallycompatible is not toxic as used, and does not have a substantiallydeleterious effect on biomolecules. Examples of negatively chargedcounterions include, among others, chloride, bromide, iodide, sulfate,alkanesulfonate, arylsulfonate, phosphate, perchlorate,tetrafluoroborate, tetraarylboride, nitrate and anions of aromatic oraliphatic carboxylic acids. Preferred counterions may include chloride,iodide, perchlorate and various sulfonates. Examples of positivelycharged counterions include, among others, alkali metal, or alkalineearth metal ions, ammonium, or alkylammonium ions.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents, which would result from writing thestructure from right to left, e.g., —CH₂O— is intended to also recite—OCH₂—.

The term “acyl” or “alkanoyl” by itself or in combination with anotherterm, means, unless otherwise stated, a stable straight or branchedchain, or cyclic hydrocarbon radical, or combinations thereof,consisting of the stated number of carbon atoms and an acyl radical onat least one terminus of the alkane radical. The “acyl radical” is thegroup derived from a carboxylic acid by removing the —OH moietytherefrom.

The term “alkyl,” by itself or as part of another substituent means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include divalent(“alkylene”) and multivalent radicals, having the number of carbon atomsdesignated (i.e. C₁-C₁₀ means one to ten carbons). Examples of saturatedhydrocarbon radicals include, but are not limited to, groups such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologsand isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, andthe like. An unsaturated alkyl group is one having one or more doublebonds or triple bonds. Examples of unsaturated alkyl groups include, butare not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl,2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and3-propynyl, 3-butynyl, and the higher homologs and isomers. The term“alkyl,” unless otherwise noted, is also meant to include thosederivatives of alkyl defined in more detail below, such as“heteroalkyl.” Alkyl groups that are limited to hydrocarbon groups aretermed “homoalkyl”.

Exemplary alkyl groups of use in the present invention contain betweenabout one and about twenty five carbon atoms (e.g. methyl, ethyl and thelike). Straight, branched or cyclic hydrocarbon chains having eight orfewer carbon atoms will also be referred to herein as “lower alkyl”. Inaddition, the term “alkyl” as used herein further includes one or moresubstitutions at one or more carbon atoms of the hydrocarbon chainfragment.

The term “amino” or “amine group” refers to the group —NR′R″ (or NRR′R″)where R, R′ and R″ are independently selected from the group consistingof hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted aryl alkyl, heteroaryl, and substituted heteroaryl. Asubstituted amine being an amine group wherein R′ or R″ is other thanhydrogen. In a primary amino group, both R′ and R″ are hydrogen, whereasin a secondary amino group, either, but not both, R′ or R″ is hydrogen.In addition, the terms “amine” and “amino” can include protonated andquaternized versions of nitrogen, comprising the group —NRR′R″ and itsbiologically compatible anionic counterions.

The term “aryl” as used herein refers to cyclic aromatic carbon chainhaving twenty or fewer carbon atoms, e.g., phenyl, naphthyl, biphenyl,and anthracenyl. One or more carbon atoms of the aryl group may also besubstituted with, e.g., alkyl; aryl; heteroaryl; a halogen; nitro;cyano; hydroxyl, alkoxyl or aryloxyl; thio or mercapto, alkyl-, orarylthio; amino, alkylamino, arylamino, dialkyl-, diaryl-, orarylalkylamino; aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl,dialkylaminocarbonyl, diarylaminocarbonyl, or arylalkylaminocarbonyl;carboxyl, or alkyl- or aryloxycarbonyl; aldehyde; aryl- oralkylcarbonyl; iminyl, or aryl- or alkyliminyl; sulfo; alkyl- oralkylcarbonyl; iminyl, or aryl- or alkyliminyl; sulfo; alkyl- orarylsulfonyl; hydroximinyl, or aryl- or alkoximinyl. In addition, two ormore alkyl or heteroalkyl substituents of an aryl group may be combinedto form fused aryl-alkyl or aryl-heteroalkyl ring systems (e.g.,tetrahydronaphthyl). Substituents including heterocyclic groups (e.g.,heteroaryloxy, and heteroaralkylthio) are defined by analogy to theabove-described terms.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a straight or branched chain, or cycliccarbon-containing radical, or combinations thereof, consisting of thestated number of carbon atoms and at least one heteroatom selected fromthe group consisting of O, N, Si, P, S, and Se and wherein the nitrogen,phosphorous, sulfur, and selenium atoms are optionally oxidized, and thenitrogen heteroatom is optionally be quaternized. The heteroatom(s) O,N, P, S, Si, and Se may be placed at any interior position of theheteroalkyl group or at the position at which the alkyl group isattached to the remainder of the molecule. Examples include, but are notlimited to, —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃,—CH₂—S—CH₂—CH₃, —CH₂—CH₂, —S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃,—Si(CH₃)₃, —CH₂—CH═N—OCH₃, and —CH═CH—N(CH₃)—CH₃. Up to two heteroatomsmay be consecutive, such as, for example, —CH₂—NH—OCH₃ and—CH₂—O—Si(CH₃)₃. Similarly, the term “heteroalkylene” by itself or aspart of another substituent means a divalent radical derived fromheteroalkyl, as exemplified, but not limited by, —CH₂—CH₂—S—CH₂—CH₂— and—CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylene groups, heteroatoms can alsooccupy either or both of the chain termini (e.g., alkyleneoxy,alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Stillfurther, for alkylene and heteroalkylene linking groups, no orientationof the linking group is implied by the direction in which the formula ofthe linking group is written. For example, the formula —C(O)₂R′—represents both —C(O)₂R′— and —R′C(O)₂—.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic moiety that can be a single ring or multiple rings (preferablyfrom 1 to 3 rings), which are fused together or linked covalently. Theterm “heteroaryl” refers to aryl groups (or rings) that contain from oneto four heteroatoms selected from N, O, S, and Se, wherein the nitrogen,sulfur, and selenium atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. A heteroaryl group can be attachedto the remainder of the molecule through a heteroatom. Non-limitingexamples of aryl and heteroaryl groups include phenyl, 1-naphthyl,2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, tetrazolyl, benzo[b]furanyl, benzo[b]thienyl,2,3-dihydrobenzo[1,4]dioxin-6-yl, benzo[1,3]dioxol-5-yl and 6-quinolyl.Substituents for each of the above noted aryl and heteroaryl ringsystems are selected from the group of acceptable substituents describedbelow.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) includes both substituted and unsubstituted forms of theindicated radical. Preferred substituents for each type of radical areprovided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) are generically referred to as “alkyl groupsubstituents,” and they can be one or more of a variety of groupsselected from, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2 m′+1), where m′ is the totalnumber of carbon atoms in such radical. R′, R″, R′″ and R″″ eachpreferably independently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, e.g., aryl substitutedwith 1-3 halogens, substituted or unsubstituted alkyl, alkoxy orthioalkoxy groups, or arylalkyl groups. When a compound includes morethan one R group, for example, each of the R groups is independentlyselected as are each R′, R″, R′″ and R″″ groups when more than one ofthese groups is present. When R′ and R″ are attached to the samenitrogen atom, they can be combined with the nitrogen atom to form a 5-,6-, or 7-membered ring. For example, —NR′R″ is meant to include, but notbe limited to, 1-pyrrolidinyl and 4-morpholinyl. From the abovediscussion of substituents, one of skill in the art will understand thatthe term “alkyl” is meant to include groups including carbon atoms boundto groups other than hydrogen groups, such as haloalkyl (e.g., —CF₃ and—CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and thelike).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are generically referredto as “aryl group substituents.” The substituents are selected from, forexample: halogen, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, -halogen,—SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl,in a number ranging from zero to the total number of open valences onthe aromatic ring system; and where R′, R″, R′″ and R″″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted aryl and substituted or unsubstituted heteroaryl. When acompound includes more than one R group, for example, each of the Rgroups is independently selected as are each R′, R″, R′″ and R″″ groupswhen more than one of these groups is present. In the schemes thatfollow, the symbol X represents “R” as described above.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CRR′)_(q)—U—, wherein T and U are independently —NR—, —O—,—CRR′— or a single bond, and q is an integer of from 0 to 3.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula -A-(CH₂)_(r)—B—, wherein A and B are independently —CRR′—, —O—,—NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′—or a single bond, and r is aninteger of from 1 to 4. One of the single bonds of the new ring soformed may optionally be replaced with a double bond. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CRR′)_(s)—X—(CR″R′″)_(d)—, where s and d are independently integers offrom 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—.The substituents R, R′, R″ and R′″ are preferably independently selectedfrom hydrogen or substituted or unsubstituted (C₁-C₆)alkyl.

As used herein, the term “heteroatom” includes oxygen (O), nitrogen (N),sulfur (S), phosphorus (P), silicon (Si), and selenium (Se).

The term “amino” or “amine group” refers to the group —NR′R″ (orN⁺RR′R″) where R, R′ and R″ are independently selected from the groupconsisting of hydrogen, alkyl, substituted alkyl, aryl, substitutedaryl, aryl alkyl, substituted aryl alkyl, heteroaryl, and substitutedheteroaryl. A substituted amine being an amine group wherein R′ or R″ isother than hydrogen. In a primary amino group, both R′ and R″ arehydrogen, whereas in a secondary amino group, either, but not both, R′or R″ is hydrogen. In addition, the terms “amine” and “amino” caninclude protonated and quaternized versions of nitrogen, comprising thegroup —N⁺RR′R″ and its biologically compatible anionic counterions.

The term “aqueous solution” as used herein refers to a solution that ispredominantly water and retains the solution characteristics of water.Where the aqueous solution contains solvents in addition to water, wateris typically the predominant solvent.

The term “Carboxyalkyl” as used herein refers to a group having thegeneral formula —(CH₂)_(n)COOH wherein n is 1-18.

The term “activated alkyne,” as used herein, refers to a chemical moietythat selectively reacts with an azide reactive group on another moleculeto form a covalent chemical bond between the activated alkyne group andthe alkyne reactive group. Examples of alkyne-reactive groups includeazides. “Alkyne-reactive” can also refer to a molecule that contains achemical moiety that selectively reacts with an alkyne group.

The term “affinity,” as used herein, refers to the strength of thebinding interaction of two molecules, such as an antibody and an antigenor a positively charged moiety and a negatively charged moiety. Forbivalent molecules such as antibodies, affinity is typically defined asthe binding strength of one binding domain for the antigen, e.g. one Fabfragment for the antigen. The binding strength of both binding domainstogether for the antigen is referred to as “avidity”. As used herein“High affinity” refers to a ligand that binds to an antibody having anaffinity constant (K_(a)) greater than 10⁴ M⁻¹, typically 10⁵-10¹¹ M⁻¹;as determined by inhibition ELISA or an equivalent affinity determinedby comparable techniques such as, for example, Scatchard plots or usingK_(d)/dissociation constant, which is the reciprocal of the K_(a).

The term “alkyne reactive,” as used herein, refers to a chemical moietythat selectively reacts with an alkyne modified group on anothermolecule to form a covalent chemical bond between the alkyne modifiedgroup and the alkyne reactive group. Examples of alkyne-reactive groupsinclude azides. “Alkyne-reactive” can also refer to a molecule thatcontains a chemical moiety that selectively reacts with an alkyne group.

The term “antibody,” as used herein, refers to a protein of theimmunoglobulin (Ig) superfamily that binds noncovalently to certainsubstances (e.g. antigens and immunogens) to form an antibody-antigencomplex. Antibodies can be endogenous, or polyclonal wherein an animalis immunized to elicit a polyclonal antibody response or by recombinantmethods resulting in monoclonal antibodies produced from hybridoma cellsor other cell lines. It is understood that the term “antibody” as usedherein includes within its scope any of the various classes orsub-classes of immunoglobulin derived from any of the animalsconventionally used.

The term “antibody fragments,” as used herein, refers to fragments ofantibodies that retain the principal selective binding characteristicsof the whole antibody. Particular fragments are well-known in the art,for example, Fab, Fab′, and F(ab′)₂, which are obtained by digestionwith various proteases, pepsin or papain, and which lack the Fc fragmentof an intact antibody or the so-called “half-molecule” fragmentsobtained by reductive cleavage of the disulfide bonds connecting theheavy chain components in the intact antibody. Such fragments alsoinclude isolated fragments consisting of the light-chain-variableregion, “Fv” fragments consisting of the variable regions of the heavyand light chains, and recombinant single chain polypeptide molecules inwhich light and heavy variable regions are connected by a peptidelinker. Other examples of binding fragments include (i) the Fd fragment,consisting of the VH and CH1 domains; (ii) the dAb fragment (Ward, etal., Nature 341, 544 (1989)), which consists of a VH domain; (iii)isolated CDR regions; and (iv) single-chain Fv molecules (scFv)described above. In addition, arbitrary fragments can be made usingrecombinant technology that retains antigen-recognition characteristics.

The term “antigen,” as used herein, refers to a molecule that induces,or is capable of inducing, the formation of an antibody or to which anantibody binds selectively, including but not limited to a biologicalmaterial. Antigen also refers to “immunogen”. The target-bindingantibodies selectively bind an antigen, as such the term can be usedherein interchangeably with the term “target”.

The term “anti-region antibody,” as used herein, refers to an antibodythat was produced by immunizing an animal with a select region that is afragment of a foreign antibody wherein only the fragment is used as theimmunogen. Regions of antibodies include the Fc region, hinge region,Fab region, etc. Anti-region antibodies include monoclonal andpolyclonal antibodies. The term “anti-region fragment” as used hereinrefers to a monovalent fragment that was generated from an anti-regionantibody of the present invention by enzymatic cleavage.

The term “aqueous solution,” as used herein, refers to a solution thatis predominantly water and retains the solution characteristics ofwater. Where the aqueous solution contains solvents in addition towater, water is typically the predominant solvent.

The term “azide reactive,” as used herein, refers to a chemical moietythat selectively reacts with an azido modified group on another moleculeto form a covalent chemical bond between the azido modified group andthe azide reactive group. Examples of azide-reactive groups includealkynes and phospines (e.g. triaryl phosphine). “Azide-reactive” canalso refer to a molecule that contains a chemical moiety thatselectively reacts with an azido group.

The term “biomolecule,” as used herein, refers to proteins, peptides,amino acids, glycoproteins, nucleic acids, nucleotides, nucleosides,oligonucleotides, sugars, oligosaccharides, lipids, hormones,proteoglycans, carbohydrates, polypeptides, polynucleotides,polysaccharides, which having characteristics typical of molecules foundin living organisms and may be naturally occurring or may be artificial(not found in nature and not identical to a molecule found in nature).

The term “buffer,” as used herein, refers to a system that acts tominimize the change in acidity or basicity of the solution againstaddition or depletion of chemical substances.

The term “carrier molecule,” as used herein, refers to a biological or anon-biological component that is covalently bonded to compound of thepresent invention. Such components include, but are not limited to, anamino acid, a peptide, a protein, a polysaccharide, a nucleoside, anucleotide, an oligonucleotide, a nucleic acid, a hapten, a psoralen, adrug, a hormone, a lipid, a lipid assembly, a synthetic polymer, apolymeric microparticle, a biological cell, a virus and combinationsthereof.

The term, “chemical handle” as used herein refers to a specificfunctional group, such as an azide, alkyne, activated alkyne, phosphite,phosphine, and the like. The chemical handle is distinct from thereactive group, defined below, in that the chemical handle are moietiesthat are rarely found in naturally-occurring biomolecules and arechemically inert towards biomolecules (e.g, native cellular components),but when reacted with an azide- or alkyne-reactive group the reactioncan take place efficiently under biologically relevant conditions (e.g.,cell culture conditions, such as in the absence of excess heat or harshreactants).

The term “click chemistry,” as used herein, refers to the Huisgencycloaddition or the 1,3-dipolar cycloaddition between an azide and aterminal alkyne to form a 1,2,4-triazole. Such chemical reactions canuse, but are not limited to, simple heteroatomic organic reactants andare reliable, selective, stereospecific, and exothermic.

The term “cycloaddition” as used herein refers to a chemical reaction inwhich two or more π (pi)-electron systems (e.g., unsaturated moleculesor unsaturated parts of the same molecule) combine to form a cyclicproduct in which there is a net reduction of the bond multiplicity. In acycloaddition, the π (pi) electrons are used to form new π (pi) bonds.The product of a cycloaddition is called an “adduct” or “cycloadduct”.Different types of cycloadditions are known in the art including, butnot limited to, [3+2] cycloadditions and Diels-Alder reactions. [3+2]cycloadditions, which are also called 1,3-dipolar cycloadditions, occurbetween a 1,3-dipole and a dipolarophile and are typically used for theconstruction of five-membered heterocyclic rings. The term “[3+2]cycloaddition” also encompasses “copperless” [3+2] cycloadditionsbetween azides and cyclooctynes and difluorocyclooctynes described byBertozzi et al. J. Am. Chem. Soc., 2004, 126:15046-15047.

The term “detectable response” as used herein refers to an occurrenceof, or a change in, a signal that is directly or indirectly detectableeither by observation or by instrumentation. Typically, the detectableresponse is an occurrence of a signal wherein the fluorophore isinherently fluorescent and does not produce a change in signal uponbinding to a metal ion or biological compound. Alternatively, thedetectable response is an optical response resulting in a change in thewavelength distribution patterns or intensity of absorbance orfluorescence or a change in light scatter, fluorescence lifetime,fluorescence polarization, or a combination of the above parameters.Other detectable responses include, for example, chemiluminescence,phosphorescence, radiation from radioisotopes, magnetic attraction, andelectron density.

The term “detectably distinct” as used herein refers to a signal that isdistinguishable or separable by a physical property either byobservation or by instrumentation. For example, a fluorophore is readilydistinguishable either by spectral characteristics or by fluorescenceintensity, lifetime, polarization or photo-bleaching rate from anotherfluorophore in the sample, as well as from additional materials that areoptionally present.

The term “directly detectable” as used herein refers to the presence ofa material or the signal generated from the material is immediatelydetectable by observation, instrumentation, or film without requiringchemical modifications or additional substances.

The term “fluorophore” as used herein refers to a composition that isinherently fluorescent or demonstrates a change in fluorescence uponbinding to a biological compound or metal ion, i.e., fluorogenic.Fluorophores may contain substitutents that alter the solubility,spectral properties or physical properties of the fluorophore. Numerousfluorophores are known to those skilled in the art and include, but arenot limited to coumarin, cyanine, benzofuran, a quinoline, aquinazolinone, an indole, a benzazole, a borapolyazaindacene andxanthenes including fluoroscein, rhodamine and rhodol as well as otherfluorophores described in RICHARD P. HAUGLAND, MOLECULAR PROBES HANDBOOKOF FLUORESCENT PROBES AND RESEARCH CHEMICALS (10^(th) edition, CD-ROM,September 2005), which is herein incorporated by reference in itsentirety.

The term “glycoprotein,” as used herein, refers to a protein that hasbeen glycosylated and those that have been enzymatically modified, invivo or in vitro, to comprise a sugar group.

The term “kit,” as used herein, refers to a packaged set of relatedcomponents, typically one or more compounds or compositions.

The term “label,” as used herein, refers to a chemical moiety or proteinthat is directly or indirectly detectable (e.g. due to its spectralproperties, conformation or activity) when attached to a target orcompound and used in the present methods, including reporter molecules,solid supports and carrier molecules. The label can be directlydetectable (fluorophore) or indirectly detectable (hapten or enzyme).Such labels include, but are not limited to, radiolabels that can bemeasured with radiation-counting devices; pigments, dyes or otherchromogens that can be visually observed or measured with aspectrophotometer; spin labels that can be measured with a spin labelanalyzer; and fluorescent labels (fluorophores), where the output signalis generated by the excitation of a suitable molecular adduct and thatcan be visualized by excitation with light that is absorbed by the dyeor can be measured with standard fluorometers or imaging systems, forexample. The label can be a chemiluminescent substance, where the outputsignal is generated by chemical modification of the signal compound; ametal-containing substance; or an enzyme, where there occurs anenzyme-dependent secondary generation of signal, such as the formationof a colored product from a colorless substrate. The term label can alsorefer to a “tag” or hapten that can bind selectively to a conjugatedmolecule such that the conjugated molecule, when added subsequentlyalong with a substrate, is used to generate a detectable signal. Forexample, one can use biotin as a tag and then use an avidin orstreptavidin conjugate of horseradish peroxidate (HRP) to bind to thetag, and then use a colorimetric substrate (e.g., tetramethylbenzidine(TMB)) or a fluorogenic substrate such as Amplex Red reagent (MolecularProbes, Inc.) to detect the presence of HRP. Numerous labels are know bythose of skill in the art and include, but are not limited to,particles, fluorophores, haptens, enzymes and their colorimetric,fluorogenic and chemiluminescent substrates and other labels that aredescribed in RICHARD P. HAUGLAND, MOLECULAR PROBES HANDBOOK OFFLUORESCENT PROBES AND RESEARCH PRODUCTS (9^(th) edition, CD-ROM,September 2002), supra.

The term “linker” or “L”, as used herein, refers to a single covalentbond or a series of stable covalent bonds incorporating 1-30 nonhydrogenatoms selected from the group consisting of C, N, O, S and P. Exemplarylinking members include a moiety that includes —C(O)NH—, —C(O)O—, —NH—,—S—, —O—, and the like. A “cleavable linker” is a linker that has one ormore cleavable groups that may be broken by the result of a reaction orcondition. The term “cleavable group” refers to a moiety that allows forrelease of a portion, e.g., a reporter molecule, carrier molecule orsolid support, of a conjugate from the remainder of the conjugate bycleaving a bond linking the released moiety to the remainder of theconjugate. Such cleavage is either chemical in nature, or enzymaticallymediated. Exemplary enzymatically cleavable groups include natural aminoacids or peptide sequences that end with a natural amino acid. Inaddition to enzymatically cleavable groups, it is within the scope ofthe present invention to include one or more sites that are cleaved bythe action of an agent other than an enzyme. Exemplary non-enzymaticcleavage agents include, but are not limited to, acids, bases, light(e.g., nitrobenzyl derivatives, phenacyl groups, benzoin esters), andheat. Many cleaveable groups are known in the art. See, for example,Jung et al., Biochem. Biophys. Acta, 761: 152-162 (1983); Joshi et al.,J. Biol. Chem., 265: 14518-14525 (1990); Zarling et al., J. Immunol.,124: 913-920 (1980); Bouizar et al., Eur. J. Biochem., 155: 141-147(1986); Park et al., J. Biol. Chem., 261: 205-210 (1986); Browning etal., J. Immunol., 143: 1859-1867 (1989). Moreover a broad range ofcleavable, bifunctional (both homo- and hetero-bifunctional) spacer armsare commercially available. An exemplary cleavable group, an ester, iscleavable group that may be cleaved by a reagent, e.g. sodium hydroxide,resulting in a carboxylate-containing fragment and a hydroxyl-containingproduct.

The term “post translational moiety” as used herein refers to any moietythat is naturally appended to a protein post translationally by arecombinant or naturally occurring enzyme. Examples include, but are notlimited to, acetate, phosphate, various lipids and carbohydrates. Asused herein “azido or alkyne modified post translational moiety” meansany post translational moiety that comprises an azido or alkyne group,which are groups rarely round in naturally occurring biological systems.

The terms “protein” and “polypeptide” are used herein in a generic senseto include polymers of amino acid residues of any length. The term“peptide” is used herein to refer to polypeptides having less than 100amino acid residues, typically less than 10 amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidues are an artificial chemical analogue of a correspondingnaturally occurring amino acid, as well as to naturally occurring aminoacid polymers.

The term “purified” as used herein refers to a preparation of aglycoprotein that is essentially free from contaminating proteins thatnormally would be present in association with the glycoprotein, e.g., ina cellular mixture or milieu in which the protein or complex is foundendogenously such as serum proteins or cellular lysate.

The term “reactive group” as used herein refers to a group that iscapable of reacting with another chemical group to form a covalent bond,i.e. is covalently reactive under suitable reaction conditions, andgenerally represents a point of attachment for another substance. Asused herein, reactive groups refer to chemical moieties generally foundin biological systems and that react under normal biological conditions,these are herein distinguished from the chemical handle, defined above,the azido and activated alkyne moieties of the present invention. Asreferred to herein the reactive group is a moiety, such as carboxylicacid or succinimidyl ester, on the compounds of the present inventionthat is capable of chemically reacting with a functional group on adifferent compound to form a covalent linkage. Reactive groups generallyinclude nucleophiles, electrophiles and photoactivatable groups.

The term “reporter molecule” refers to any moiety capable of beingattached to a modified post translationally modified protein of thepresent invention, and detected either directly or indirectly. Reportermolecules include, without limitation, a chromophore, a fluorophore, afluorescent protein, a phosphorescent dye, a tandem dye, a particle, ahapten, an enzyme and a radioisotope. Preferred reporter moleculesinclude fluorophores, fluorescent proteins, haptens, and enzymes.

The term “sample” as used herein refers to any material that may containan analyte for detection or quantification or a modified posttranslationally modified protein of the present invention. The analytemay include a reactive group, e.g., a group through which a compound ofthe invention can be conjugated to the analyte. The sample may alsoinclude diluents, buffers, detergents, and contaminating species, debrisand the like that are found mixed with the target. Illustrative examplesinclude urine, sera, blood plasma, total blood, saliva, tear fluid,cerebrospinal fluid, secretory fluids from nipples and the like. Alsoincluded are solid, gel or sol substances such as mucus, body tissues,cells and the like suspended or dissolved in liquid materials such asbuffers, extractants, solvents and the like. Typically, the sample is alive cell, a biological fluid that comprises endogenous host cellproteins, nucleic acid polymers, nucleotides, oligonucleotides, peptidesand buffer solutions. The sample may be in an aqueous solution, a viablecell culture or immobilized on a solid or semi solid surface such as apolyacrylamide gel, membrane blot or on a microarray.

The term “solid support,” as used herein, refers to a material that issubstantially insoluble in a selected solvent system, or which can bereadily separated (e.g., by precipitation) from a selected solventsystem in which it is soluble. Solid supports useful in practicing thepresent invention can include groups that are activated or capable ofactivation to allow selected one or more compounds described herein tobe bound to the solid support.

The term “Staudinger ligation” as used herein refers to a chemicalreaction developed by Saxon and Bertozzi (E. Saxon and C. Bertozzi,Science, 2000, 287: 2007-2010) that is a modification of the classicalStaudinger reaction. The classical Staudinger reaction is a chemicalreaction in which the combination of an azide with a phosphine orphosphite produces an aza-ylide intermediate, which upon hydrolysisyields a phosphine oxide and an amine. A Staudinger reaction is a mildmethod of reducing an azide to an amine; and triphenylphosphine iscommonly used as the reducing agent. In a Staudinger ligation, anelectrophilic trap (usually a methyl ester) is appropriately placed onthe aryl group of a triarylphosphine (usually ortho to the phosphorusatom) and reacted with the azide, to yield an aza-ylide intermediate,which rearranges in aqueous media to produce a compound with amide groupand a phosphine oxide function. The Staudinger ligation is so namedbecause it ligates (attaches/covalently links) the two startingmolecules together, whereas in the classical Staudinger reaction, thetwo products are not covalently linked after hydrolysis.

The terms “structural integrity of the [biomolecule] is not reduced” or“preservation of the structural integrity of the [biomolecule]”, as usedherein, means that either: 1) when analyzed by gel electrophoresis anddetection (such as staining), a band or spot arising from the labeledbiomolecule is not reduced in intensity by more than 20%, and preferablynot reduced by more than 10%, with respect to the corresponding band orspot arising from the same amount of the electrophoresed unlabeledbiomolecule, arising from the labeled biomolecule analyzed; or 2) whenanalyzed by gel electrophoresis, a band or spot arising from the labeledbiomolecule is not observed to be significantly less sharp than thecorresponding band or spot arising from the same amount of theelectrophoresed unlabeled biomolecule, where “significantly less sharp”(synonymous with “significantly more diffuse”) means the detectable bandor spot takes up at least 5% more, preferably 10% more, more preferably20% more area on the gel than the corresponding unlabeled biomolecule.Other reproducible tests for structural integrity of labeledbiomolecules include, without limitation detection of released aminoacids or peptides, or mass spectrometry.

In general, for ease of understanding the present invention, themetabolic and enzymatic labeling of biomolecules with azide moieties,alkyne moieties or phosphine, and the chemical labeling of such moietieswith azide reactive moieties, alkyne reactive moieties or phosphinereactive moieties will first be described in detail. This will befollowed by some embodiments in which such labeled biomolecules can bedetected, isolated and/or analyzed. Exemplified methods are thendisclosed.

Modification of Biomolecules

The tagging/labeling of biomolecules, including glycoproteins,phosphoproteins, isoprenylated proteins, can utilize variouspost-translation modifications to incorporate a bioorthoganol moietyinto a biomolecule followed by chemical attachment of a label (reportermolecule, solid support, and carrier molecule). An alternative approachis to incorporate a bioorthoganol moiety into the biomolecule usingcellular biosynthetic pathways, or metabolic modifications. Thesebioorthogonol moieties are non-native, non-perturbing chemical handlespossessing unique chemical functionality that can be modified throughhighly selective reactions. Examples of such moieties include, but arenot limited to hydrazide and aminooxy derivatives, azides that can beselectively modified with phosphines (Staudinger ligation), azides thatcan be selectively modified with activated alkynes, and azides that canbe selectively modified with terminal alkynes (“click” chemistry).

Post-translational modification is alteration of a primary structure ofthe protein after the protein has been translated. After translation,the post-translational modification of amino acids extends the range offunctions of the protein by attaching to it other biochemical functionalgroups such as acetate, phosphate, various lipids and carbohydrates. Inaddition, the range of functions of proteins can be extended bypost-translational modifications that change the chemical nature of anamino acid or by making structural changes such as disulfide bridgesformation. Other post-translational modifications involve enzymes thatremove amino acids from the amino end (N-terminus) of the protein, orcut the protein chain. Post-translational modifications act onindividual residues either by cleavage at specific points, deletions,additions or by converting or modifying side chains.

The various post-translational modifications that can be used with themethods and compositions described herein include, but are not limitedto, cleavage, N-terminal extensions, protein degradation, acylation ofthe N-terminus, biotinylation (acylation of lysine residues with abiotin), amidation of the C-terminal, glycosylation, iodination,covalent attachment of prosthetic groups, acetylation (the addition ofan acetyl group, usually at the N-terminus of the protein), alkylation(the addition of an alkyl group (e.g. methyl, ethyl, propyl) usually atlysine or arginine residues), methylation, adenylation,ADP-ribosylation, covalent cross links within, or between, polypeptidechains, sulfonation, prenylation, Vitamin C dependent modifications(proline and lysine hydroxylations and carboxy terminal amidation),Vitamin K dependent modification wherein Vitamin K is a cofactor in thecarboxylation of glutamic acid residues resulting in the formation ofa-carboxyglutamate (a gla residue), glutamylation (covalent linkage ofglutamic acid residues), glycylation (covalent linkage glycineresidues), glycosylation (addition of a glycosyl group to eitherasparagine, hydroxylysine, serine, or threonine, resulting in aglycoprotein), isoprenylation (addition of an isoprenoid group such asfarnesol and geranylgeraniol), lipoylation (attachment of a lipoatefunctionality), phosphopantetheinylation (addition of a4′-phosphopantetheinyl moiety from coenzyme A, as in fatty acid,polyketide, non-ribosomal peptide and leucine biosynthesis),phosphorylation (addition of a phosphate group, usually to serine,tyrosine, threonine or histidine), and sulfation (addition of a sulfategroup, usually to a tyrosine residue). The post-translationalmodifications that change the chemical nature of amino acids include,but are not limited to, citrullination (the conversion of arginine tocitrulline by deimination), and deamidation (the conversion of glutamineto glutamic acid or asparagine to aspartic acid). The post-translationalmodifications that involve structural changes include, but are notlimited to, formation of disulfide bridges (covalent linkage of twocysteine amino acids) and proteolytic cleavage (cleavage of a protein ata peptide bond). Certain post-translational modifications involve theaddition of other proteins or peptides, such as ISGylation (covalentlinkage to the ISG15 protein (Interferon-Stimulated Gene)), SUMOylation(covalent linkage to the SUMO protein (Small Ubiquitin-relatedMOdifier)) and ubiquitination (covalent linkage to the proteinubiquitin). In certain embodiments, selenoproteins are used in themethods and compositions described herein.

Glycoproteins are biomolecules composed of proteins covalently linked tocarbohydrates. Certain post-translational modifications append a sugarmoiety (carbohydrate or oligosaccharide) onto a protein, thereby forminga glycoprotein. The common monosaccharides found in glycoproteinsinclude, but are not limited to, glucose, galactose, mannose, fucose,xylose, N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc) andN-acetylneuraminic acid (NANA, also known as sialic acid). In additionthe sugar moiety can be a glycosyl group. In glycoproteins thecarbohydrates can be linked to the protein component by eitherN-glycosylation or O-glycosylation. N-glycosylation commonly occursthrough asparagine forming an N-glycosidic linkage via an amide group.O-glycosylation commonly occurs at hydroxylysine, hydroxyproline,serine, or threonine, forming an O-glycosidic linkage.

The metabolic labeling of glycoproteins with azido sugars has beendescribed, however these currently known methods lack sufficientmethodology for detecting and isolating such azido modifiedglycoproteins. Provided herein are methods and compositions for thedetection, isolation and/or analysis of glycosylated proteinsfacilitated by the incorporation and use of unnatural azido sugars intoglycoproteins rather than natural sugars. In particular, presented are anovel methods for A) labeling azido modified biomolecules in solutionfollowed by separation using methods known in the art for separatingbiomolecules based on size, weight and/or charge, B) labelingimmobilized azido modified biomolecules and C) novel methods forenzymatically labeling a biomolecule with an azide group. These azidomodified biomolecules can form conjugates with reporter molecules,carrier molecules or solid supports, provided an azide reactive group ispresent, using either methods known in the art or the current methodsprovided herein.

Also provided herein are methods and compositions for the detection,isolation and/or analysis of glycosylated proteins facilitated by theincorporation and use of unnatural alkyne containing sugars intoglycoproteins rather than natural sugars. In particular, presented are anovel methods for A) labeling alkyne modified biomolecules in solutionfollowed by separation using methods known in the art for separatingbiomolecules based on size, weight and/or charge, B) labelingimmobilized alkyne modified biomolecules and C) novel methods forenzymatically labeling a biomolecule with an alkyne group. These alkynemodified biomolecules can form conjugates with reporter molecules,carrier molecules or solid supports, that have azide groups, usingeither methods known in the art or the current methods provided herein.

The methods described herein can be used for metabolic and enzymaticselective labeling of different subclasses of glycoproteins, includingcell surface N- and O-linked glycoproteins and intracellularO-GlcNAc-modified proteins. Such labeling enables highly-sensitivedetection of labeled glycoproteins using chromatographic andelectrophoretic techniques including, but not limited to, gelelectrophoresis and western blot analysis. (Dube D. H., Bertozzi C. R.(2003). Curr Opin Chem Biol. October; 7(5):616-25; Boeggeman E. E.,Ramakrishnan B., Qasba P. K. (2003). Protein Expr Purif August;30(2):219-29; Khidekel N., Arndt S., Lamarre-Vincent N., Lippert A.,Poulin-Kirstien K. G., Ramakrishnan B., Qasba P. K., Hsieh-Wilson L. C.(2003). J Am Chem Soc December 31; 125(52):16162-3). In certainembodiments, glycoproteins are “labeled” with azide modified sugarmoieties, while in other embodiments glycoproteins are “labeled” withalkyne modified sugar moieties. This can be accomplished eithermetabolically, wherein cells are fed either unnatural azide containingsugars or alkyne containing sugars, or it is accomplished in vitro byenzymatic methods. In a specific embodiment of such enzymatic methods,the galactose-1-phosphate uridyl transferase (GalT) enzyme is used toincorporate a UDP-GalNAz moiety to a protein.

Phosphoproteins are biomolecules composed of proteins having a phosphategroup covalently linked to one or more serine, threonine, or tyrosineresidues. Phosphate groups are added to proteins post translationally bykinases and removed by phosphorylases; changes in the phosphorylationstate of a protein can have a significant effect on its structure andfunction.

Phosphoproteins and phosphopeptides can also be modified by the additionof an azide or alkyne group. For example, a one or more phosphate groupson a phosphoprotein can be converted to an azido or alkyne, by usingbase treatment to remove the phosphate group of phosphoserine,converting it to dehydroamino-2-butyric acid, or to remove the phosphategroup of threonine to convert it to dehydroalanine. The dephosphorylatedprotein can then be reacted with a thiol or amine-containing compoundthat also comprises an azide or terminal alkyne to form an azido oralkyne labeled (phospho)protein. A phosphoprotein so modified isreferred to herein as a modified phosphoproteins, although all phosphategroups of the modified phosphoprotein may have been removed.

The invention thus includes a method of modifying a phosphoprotein toinclude an alkyne or azido group, in which the method includes:contacting a phosphoprotein that includes at least one phosphoserineresidue or at least one phosphothreonine residue with a base solutionthat removes phosphates from threonine and serine residues of thephosphorylated protein to form a protein comprising at least onedehydroalanine or at least one dehydroamino-2-butyric acid; contactingthe dehydroalanine or dehydroamino-2-butyric acid with a compound thatincludes a thio or amine group and has an azide or terminal alkyne toform an azido or alkyne modified protein; and contacting the azido orterminal alkyne-modified protein with a reporter molecule, carriermolecule, or solid support that comprises an azido moiety, where thephosphoprotein is labeled with a terminal alkyne, or a reportermolecule, carrier molecule, or solid support that comprises an alkyne,where the phosphoprotein is modified to have an azido group. Themodified phosphoproteins comprising azido or alkyne groups can be usedin the labeling methods provided herein.

Metabolic Modification

The modified proteins used in the methods and compostions describedherein can be formed by in vivo metabolic modification. Such in vivometabolic modification of proteins can be accomplished using the methodsdescribed in U.S. Pat. No. 6,936,701. In general, cells are fednon-natural sugars having a desired functional group including, but notlimited to, azide moieties and alkyne moieties and phosphine moieties.These non-natural sugars are then attached to the protein forming aglycoprotein conjugate which is then expressed by the cell. The modifiedproteins are either naturally secreted and isolated, or they can bepresent, for example, in a cell lysate, extracellular milieu, cellfraction, or isolated from a complex protein mixture. The resultingmodified protein is then used in the methods and compositions describedherein.

In certain embodiments, such non-natural sugars contain a moiety thatfacilitates entry into the cell including, but not limited to, atetraacetyl moiety. Thus, non-natural sugar substrates, used in themetabolic labeling of proteins used in the methods and compositionsdescribed herein, include small groups that the cellular machinery wouldbe more likely to incorporate, and not recognize as being foreign. Thecellular metabolic machinery incorporates the substrates into N- orO-linked glycans attached to the proteins.

One aspect of the methods provided herein involves metabolicallylabelling proteins with non-natural sugar substrates that have azidogroups or alkyne groups that may be used for “click” chemistry asdescribed herein. Another aspect of the methods provided herein involvesmetabolically labelling proteins with non-natural sugar substrates thathave azido groups or phosphine groups that may be used in a Staudingerligation as described herein. Such non-natural sugars can be synthesizedusing methods known in the art. In certain embodiments, the non-naturalsugars used to metabolically label proteins are non-naturalazido-containing sugars including, but are not limited to, GlcNAz,GalNAz and ManNAz, or tetraacetylated non-natural azido-containingsugars including, but not limited to, GlcNAz, ManNAz, and GalNAz. Incertain embodiments, the non-natural sugars used to metabolically labelglycoproteins or specific subsets of cellular glycoproteins in culturedcells are non-natural azido-containing sugars including, but are notlimited to, GlcNAz, GalNAz and ManNAz, or tetraacetylated non-naturalazido-containing sugars including, but not limited to, GlcNAz, ManNAz,and GalNAz. Certain embodiments of metabolic labeling with non-naturalazido-containing sugars is shown in FIG. 1, and described in Example 1,wherein cells are fed non-natural azido-containing sugars to obtainmodified proteins, including modified glycoproteins, used in the methodsand compositions described herein. FIG. 2 shows gel images of modifiedproteins obtained from Jurkat cells which were fed Ac₄ManNAz orAc₄GalNAz for 3 days (A) or Ac₄GlcNAz overnight (B). The modifiedproteins were obtained from harvested cells and labeled with afluorescent alkyne probe using “click” chemistry (C). The gels were thenstained with a protein stain that has different excitation/emissionproperties than the alkyne probe, thereby demonstrating the selectivityof “click” chemistry for glycoprotein labeling.

Proteins metabolically labeled with non-natural sugars, followed bylabeling using “click” chemistry or Staudinger ligation, can be detectedusing 2-D gel electrophoresis. FIG. 3 shows a 2-D gel separation ofproteins obtained from soluble Jurkat cell treated with Ac₄GlcNAz andlabeled with a fluorescent alkyne probe using click chemistry. Thecontrol cell culture was not fed Ac₄GlcNAz and therefore does not showthe presence of the fluorescent alkyne probe. However, staining with aprotein stain shows the presence of protein in both gels.

The unnatural sugars described herein can be incorporated into variousglycoproteins and subclasses of glycoproteins. In certain embodiments,the glycoproteins are antibodies from antibody-producing cells such as,by way of example only, hybridoma cells, and any other cell thatproduces antibodies or recombinant antibodies. Once the in vivo labeledantibodies are released and isolated from the cells, the labeledantibodies may be directly labeled using an azide reactive reportermolecule, solid support or carrier molecule, as described herein. Thereporter molecules can include, but are not limited to labels, while thesolid supports can include, but are not limited to, solid supportresins, microtiter plates and microarray slides. The carrier moleculescan include, but are not limited to, affinity tags, nucleotides,oligonucleotides and polymers.

Enzymatic Modification

The modified proteins used in the methods and compositions describedherein can be modified in vitro using enzymatic post-translationalmodification. In certain embodiments the post-translationalmodifications used in the methods and compostions described hereininclude, but are not limited to, glycosylation, isoprenylation,lipoylation and phosphorylation. In certain embodiments, suchpost-translational modifications are used to modify proteins with azidemoieties, alkyne moieties, or phosphine moieties. Such phosphinemoieties include, but are not limited to, triaryl phosphines. Certainembodiments utilize β-1,4-galactosyltransferase(GalT), or a mutantthereof, to modify glycosylated proteins with azide moieties, alkynemoieties, or phosphine moieties. β-1,4-galactosyltransferase (GalT) isan enzyme that can catalyze the transfer of galactose from uridinediphosphate-GalNAz (UDP-GalNAz) to terminal GlcNAc groups. Thus,glycoproteins are enzymatically labeled in vitro with azide modifiedsugar moieties, alkyne modified sugar moieties, or phosphine modifiersugar moieties. In another embodiment, GalT has been mutated, such aswith a single Y289L mutation, to enlarge the binding pocket and toenhance the catalytic activity toward substrates. Other mutations toGalT are contemplated such that the mutation provide enlargement of thebinding pocket and enhancement of the catalytic activity towardsubstrates. As shown in FIGS. 6A-B, a mutant of β-GalT enzyme (β-GalT1)can be used to enzymatically label an O-GlcNAc containing protein withazide (UDP-GalNAz). In addition FIG. 6A-B show that UDP-GalNAz is also asuitable substrate for the mutant of -1,4-galactosyltransferase (GalT1)resulting in the ability to enzymatically attach an non-naturalazido-containing sugar to a glycoprotein.

The methods and compositions described herein for enzymatically labelingglycoproteins with azido-containing sugars, alkyne-containing sugars orphosphine-containing sugars provide for the rapid, selective andsensitive detection of post-translationally modified proteins,including, but not limited to, those with post-translationalglycosylations. Such detection methods utilize labeling using theselective reactivity of “click” chemistry, or Staudinger ligation, asdescribed herein. Such labeling methods can be used to detectpost-translational modifications on proteins in which such modificationswere undetectable using other techniques. In certain embodiments, suchlabeling methods can be used to detect O-GlcNAc post-translationalmodifications on proteins in which such modifications are undetectableusing other techniques.

In certain embodiments, enzymatic post-translational modificationmethods are used to selectively transfer azido, alkyne of phosphinefunctionality onto proteins using azido-containing substrates,alkyne-containing substrates or phosphine-containing substrates. Oncetransferred the modified protein can then be conjugated to a reportermolecule, solid support or carrier, molecule using a “click” chemistry,Staudinger ligation, or activated alkyne based reactions, therebyenabling detection, isolation and/or analysis of the modified protein.Thus, in certain embodiments, the in vitro enzymatic labeling allows forsensitive detection of modified proteins from cells or tissues.

In certain embodiments, such methods are used to exploit the ability ofan engineered mutant of -1,4-galactosyltransferase to selectivelytransfer azido functionality onto O-GlcNAc glycosylated proteins usingnon-natural azido-containing sugars, alkyne functionality onto O-GlcNAcglycosylated proteins using non-natural alkyne-containing sugars, orphosphine functionality onto O-GlcNAc glycosylated proteins usingnon-natural phosphine-containing sugars. Once transferred the azidomodified glycoprotein, alkyne modified glycoprotein or phosphinemodified glycoprotein can then be conjugated to a reporter molecule,solid support or carrier molecule using a “click” chemistry, Staudingerligation or activated alkyne based reactions, thereby enablingdetection, isolation and/or analysis of the modified protein. Thus, incertain embodiments, the in vitro labeling with the modified-GalT1enzyme allows for sensitive detection of O-GlcNAc-modified proteins fromcells or tissues and enables the characterization of inverserelationships between cellular O-GlcNAc modification and phosphorylationon the same proteins.

The methods and compositions described herein can be used toenzymatically label antibodies in vitro with azido-containing sugars,alkyne-containing sugars or phosphine-containing sugars can be used tolabel antibodies. Once the in vitro labeled antibodies are obtained, thelabeled antibodies may be directly labeled using either an azidereactive, alkyne reactive or phosphine reaction reporter molecule, solidsupport or carrier molecule, as described herein. In certainembodiments, as shown in FIG. 11, antibodies can be enzymaticallylabeled with UDPGalNAz using GalT via reaction of the terminal OGlcNAcmolecules present on antibody carbohydrates. “Click” chemistry orStaudinger ligation can then be used to conjugate a dye or a hapten,such as biotin, (or other reporter molecules, solid supports or carriermolecules) to the attached azide moiety. In certain embodiments, if noOGlcNAc sugars are present on an antibody, then use of the Endo-H(endo-β-N-acetylglucosaminidase H) enzyme will be used to generate atruncated chain which terminates with one N-acetylglucosamine residue(FIG. 11B). FIG. 17 shows the enzymatic labeling of Goat antibody withUDP-GalNAz followed by conjugation with an alkyne-TAMRA dye using“click: chemistry. The selectivity and specificity of such conjugationsis shown from the controls and general protein staining with a SYPRO®Ruby protein stain.

The invention also includes methods for enzymatically labeling proteinsvia attachment of an azido or alkyne modified phosphate to the protein.Kinases that phosphorylate serine, threonine, and also tyrosine residuesare known in the art and can be used for the attachment of azido oralkyne groups to a protein. Included herein is a method for forming aphosphoprotein comprising a terminal alkyne modified phosphate thatincludes contacting a protein with a terminal alkyne modified phosphatein the presence of an enzyme that will transfer the terminal alkynemodified phosphate to the protein to form a terminal alkyne modifiedphosphoprotein. In some embodiments, the protein has at least oneserine, threonine, or tyrosine residue. The invention also includesmethods for forming a phosphoprotein conjugate, in which thephosphoprotein conjugate is attached to a reporter molecule, solidsupport, or carrier molecule, in which the method includes contacting aprotein with a terminal alkyne modified phosphate in the presence of anenzyme that will transfer the terminal alkyne modified phosphate to theprotein to form a terminal alkyne modified phosphoprotein; andcontacting the terminal alkyne modified phosphoprotein with a reportermolecule, carrier molecule or solid support that comprises an azidomoiety to form phosphoprotein reporter molecule, carrier molecule orsolid support conjugate.

Also included herein is a method for forming a phosphoprotein comprisinga terminal azido modified phosphate that includes contacting a proteinwith an azido modified phosphate in the presence of an enzyme that willtransfer the azido modified phosphate to the protein to form an azidomodified phosphoprotein. In some embodiments, the protein has at leastone serine, threonine, or tyrosine residue. The invention also includesmethods for forming a phosphoprotein conjugate, in which thephosphoprotein conjugate is attached to a reporter molecule, solidsupport, or carrier molecule, in which the method includes contacting aprotein with an azido modified phosphate in the presence of an enzymethat will transfer the azido modified phosphate to the protein to forman azido modified phosphoprotein; and contacting the azido modifiedphosphoprotein with a reporter molecule, carrier molecule or solidsupport that comprises a terminal alkyne moiety to form phosphoproteinreporter molecule, carrier molecule or solid support conjugate.

The methods described herein can also be used to label proteins whichhave been modified with lipids by post-translational lipoylationincluding, but not limited to, palmitoylation and myristolation. In suchpost-translational modifications azide-containing lipids,alkyne-containing lipids or phosphine-containing lipids are used totransfer azide moieties, alkyne moieties or phosphine moieties toproteins, whereupon such moieties are used to label the modified proteinwith a reporter molecule, carrier molecule and/or solid substrate lysingthe methods described herein. An example of such labeling of proteins isgiven in Example 32 and Example 33.

The methods described herein can be used to label proteins which havebeen modified with isoprenoid groups by post-translationalisoprenylation including, but not limited to, farnesylation andgeranylgeranylation. In such post-translational modificationsazide-containing isoprenoids, alkyne-containing isoprenoids orphosphine-containing isoprenoids are used to transfer azide moieties,alkyne moieties or phosphine moieties to proteins, whereupon suchmoieties are used to label the modified protein with a reportermolecule, carrier molecule and/or solid substrate using the methodsdescribed herein. An example of such labeling of proteins is given inExample 31.

Chemical Modification of Biomolecules Containing Azide, Alkyne orPhosphine Moieties

Biomolecules that can be chemically modified using the methods describedherein include, but are not limited to, proteins, peptides, amino acids,glycoproteins, nucleic acids, nucleotides, nucleosides,oligonucleotides, sugars, oligosaccharides, lipids, hormones,proteoglycans, carbohydrates, polypeptides, polynucleotides andpolysaccharides. Such biomolecules can contain azide moieties, alkynemoieties or phosphine moieties that are incorporated into biomoleculesusing post-translational modifications via cellular biosyntheticpathways (metabolic labeling) or enzymatic labeling as described herein.Alternatively, azide moieties, alkyne moieties or phosphine moieties canbe incorporated into biomolecules using chemical synthetic procedures asdescribed herein. These azide moieties, alkyne moieties and phosphinemoieties are non-native, non-perturbing bioorthogonol chemical moietiesthat possess unique chemical functionality that can be modified throughhighly selective reactions. Such reactions used in the methods describedherein, wherein the chemical modification of biomolecules that containazide moieties or alkyne moieties utilize Copper(I)-catalyzedAzide-Alkyne Cycloaddition, also referred to herein as “click”chemistry, the chemical modification of biomolecules that contain azidemoieties or phosphine moieties utilize Staudinger ligation, and thechemical modification of biomolecules that contain activated-alkynemoieties or activated-alkyne reactive moieties.

In certain embodiments, the biomolecules used in the methods andcompositions described herein can be labeled chemically, enzymatically(for example, by enzymatic in vitro incorporation into the biomoleculeof a moiety that includes an azido group or terminal alkyne), or bysupplying cells with alkyne or azido-containing molecular precursors(e.g., amino acids or sugars) that can be incorporated into biomoleculesin vivo. Such methods are described herein.

“Click” Chemistry

Azides and terminal or internal alkynes can undergo a 1,3-dipolarcycloaddition (Huisgen cycloaddition) reaction to give a 1,2,3-triazole.However, this reaction requires long reaction times and elevatedtemperatures. Alternatively, azides and terminal alkynes can undergoCopper(I)-catalyzed Azide-Alkyne Cycloaddition (CuAAC) at roomtemperature. Such copper(I)-catalyzed azide-alkyne cycloadditions, alsoknown as “click” chemistry, is a variant of the Huisgen 1,3-dipolarcycloaddition wherein organic azides and terminal alkynes react to give1,4-regioisomers of 1,2,3-triazoles. Examples of “click” chemistryreactions are described by Sharpless et al. (U.S. Patent ApplicationPublication No. 20050222427, published Oct. 6, 2005, PCT/US03/17311;Lewis W G, et al., Angewandte Chemie-Int'l Ed. 41 (6): 1053; methodreviewed in Kolb, H. C., et al., Angew. Chem. Inst. Ed. 2001,40:2004-2021), which developed reagents that react with each other inhigh yield and with few side reactions in a heteroatom linkage (asopposed to carbon-carbon bonds) in order to create libraries of chemicalcompounds. As described herein, “click” chemistry is used in the methodsfor labeling biomolecules.

The copper used as a catalyst for the “click” chemistry reaction used inthe methods described herein to conjugate a label to a biomolecule is inthe Cu (I) reduction state. The sources of copper(I) used in suchcopper(I)-catalyzed azide-alkyne cycloadditions can be any cuprous saltincluding, but not limited to, cuprous halides such as cuprous bromideor cuprous iodide. However, this regioselective cycloaddition can alsobe conducted in the presence of a metal catalyst and a reducing agent.In certain embodiments, copper can be provided in the Cu (II) reductionstate (for example, as a salt, such as but not limited to Cu(NO₃)₂Cu(OAc)₂ or CuSO₄), in the presence of a reducing agent wherein Cu(I) isformed in situ by the reduction of Cu(II). Such reducing agents include,but are not limited to, ascorbate, Tris(2-Carboxyethyl) Phosphine(TCEP), 2,4,6-trichlorophenol (TCP), NADH, NADPH, thiosulfate, metalliccopper, quinone, hydroquinone, vitamin K₁, glutathione, cysteine,2-mercaptoethanol, dithiothreitol, Fe²⁺, Co²⁺, or an applied electricpotential. In other embodiments, the reducing agents include metalsselected from Al, Be, Co, Cr, Fe, Mg, Mn, Ni, Zn, Au, Ag, Hg, Cd, Zr,Ru, Fe, Co, Pt, Pd, Ni, Rh, and W.

The copper(I)-catalyzed azide-alkyne cycloadditions for labelingbiomolecules can be performed in water and a variety of solvents,including mixtures of water and a variety of (partially) miscibleorganic solvents including alcohols, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), tert-butanol (tBuOH) and acetone.

Without limitation to any particular mechanism, copper in the Cu (I)state is a preferred catalyst for the copper(I)-catalyzed azide-alkynecycloadditions, or “click” chemistry reactions, used in the methodsdescribed herein. Certain metal ions are unstable in aqueous solvents,by way of example Cu(I), therefore stabilizing ligands/chelators can beused to improve the reaction. In certain embodiments at least one copperchelator is used in the methods described herein, wherein such chelatorsbinds copper in the Cu (I) state. In certain embodiments at least onecopper chelator is used in the methods described herein, wherein suchchelators binds copper in the Cu (II) state. In certain embodiments, thecopper (I) chelator is a 1,10 phenanthroline-containing copper (I)chelator. Non-limiting examples of such phenanthroline-containing copper(I) chelators include, but are not limited to, bathophenanthrolinedisulfonic acid (4,7-diphenyl-1,10-phenanthroline disulfonic acid) andbathocuproine disulfonic acid (BCS;2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline disulfonate). Otherchelators used in such methods include, but are not limited to,N-(2-acetamido)iminodiacetic acid (ADA), pyridine-2,6-dicarboxylic acid(PDA), S-carboxymethyl-L-cysteine (SCMC), trientine,tetra-ethylenepolyamine (TEPA),N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), EDTA,neocuproine, N-(2-acetamido)iminodiacetic acid (ADA),pyridine-2,6-dicarboxylic acid (PDA), S-carboxymethyl-L-cysteine (SCMC),tris-(benzyl-triazolylmethyl)amine (TBTA), or a derivative thereof. Mostmetal chelators, a wide variety of which are known in the chemical,biochemical, and medical arts, are known to chelate several metals, andthus metal chelators in general can be tested for their function in 1,3cycloaddition reactions catalyzed by copper. In certain embodiments,histidine is used as a chelator, while in other embodiments glutathioneis used as a chelator and a reducing agent.

The concentration of the reducing agents used in the “click” chemistryreaction described herein can be in the micromolar to millimolar range.In certain embodiments the concentration of the reducing agent is fromabout 100 micromolar to about 100 millimolar. In other embodiments theconcentration of the reducing agent is from about 10 micromolar to about10 millimolar. In other embodiments the concentration of the reducingagent is from about 1 micromolar to about 1 millimolar.

In certain embodiments, the methods describe herein for labelingbiomolecules using “click” chemistry, at least one copper chelator isadded after copper(II) used in the reaction has been contacted with areducing agent. In other embodiments, at least one copper chelator canbe added immediately after contacting copper(II) with a reducing agent.In other embodiments, the copper chelator(s) is added between about fiveseconds and about twenty-four hours after copper(II) and a reducingagent have been combined in a reaction mixture. In other embodiments, atleast one copper chelator can be added any time to a reaction mixturethat includes copper(II) and a reducing agent, such as, by way ofexample only, immediately after contacting copper(II) and a reducingagent, or within about five minutes of contacting copper(II) and areducing agent in the reaction mixture. In some embodiments, at leastone copper chelator can be added between about five seconds and aboutone hour, between about one minute and about thirty minutes, betweenabout five minutes and about one hour, between about thirty minutes andabout two hours, between about one hour and about twenty-four hours,between about one hour and about five hours, between about two hours andabout eight hours, after copper(II) and a reducing agent have beencombined for use in a reaction mixture.

In other embodiments, one or more copper chelators can be added morethan once to such “click” chemistry reactions. In embodiments in whichmore than one copper chelators is added to a reaction, two or more ofthe copper chelators can bind copper in the Cu (I) state or, one or moreof the copper chelators can bind copper in the Cu (I) state and one ormore additional chelators can bind copper in the Cu (II) state. Incertain embodiments, one or more copper chelators can be added after theinitial addition of a copper chelator to the “click” chemistry reaction.In certain embodiments, the one or more copper chelators added after theinitial addition of a copper chelator to the reaction can be the same ordifferent from a copper chelator added at an earlier time to thereaction.

The concentration of a copper chelator used in the “click” chemistryreaction described herein can be determined and optimized using methodswell known in the art, including those disclosed herein using “click”chemistry to label biomolecules followed by detecting such labeledbiomolecules to determine the efficiency of the labeling reaction andthe integrity of the labeled biomolecule(s) (see FIG. 5). In certainembodiments, the chelator concentrations used in the methods describedherein is in the micromolar to millimolar range, by way of example only,from 1 micromolar to 100 millimolar. In certain embodiments the chelatorconcentration is from about 10 micromolar to about 10 millimolar. Inother embodiments the chelator concentration is from about 50 micromolarto about 10 millimolar. In other embodiments the chelator, can beprovided in a solution that includes a water miscible solvent such as,alcohols, dimethyl sulfoxide (DMSO), dimethyl formamide (DMF),tert-butanol (tBuOH) and acetone. In other embodiments the chelator, canbe provided in a solution that includes a solvent such as, for example,dimethyl sulfoxide (DMSO) or dimethylformamide (DMF).

In certain embodiments of the methods for labeling biomolecule utilizing“click” chemistry described herein, the biomolecule can possess an azidemoiety, whereupon the label possesses an alkyne moiety, whereas in otherembodiments the biomolecule can possess an alkyne moiety, and the labelpossesses an azide moiety.

Staudinger Ligation

The Staudinger reaction, which involves reaction between trivalentphosphorous compounds and organic azides (Staudinger et al. Helv. Chim.Acta 1919, 2, 635), has been used for a multitude of applications.(Gololobov et al. Tetrahedron 1980, 37, 437); (Gololobov et al.Tetrahedron 1992, 48, 1353). There are almost no restrictions on thenature of the two reactants. The Staudinger ligation is a modificationof the Staudinger reaction in which an electrophilic trap (usually amethyl ester) is placed on a triaryl phosphine. In the Staudingerligation, the aza-ylide intermediate rearranges, in aqueous media, toproduce an amide linkage and the phosphine oxide, ligating the twomolecules together, whereas in the Staudinger reaction the two productsare not covalently linked after hydrolysis. Such ligations have beendescribed in U.S. Patent Application No. 20060276658. In certainembodiments, the phosphine can have a neighboring acyl group such as anester, thioester or N-acyl imidazole (i.e. a phosphinoester,phosphinothioester, phosphinoimidazole) to trap the aza-ylideintermediate and form a stable amide bond upon hydrolysis. In certainembodiments, the phosphine can be a di- or triarylphosphine to stabilizethe phosphine. The phosphines used in the Staudinger ligation methodsdescribed herein to conjugate a label to a biomolecule include, but arenot limited to, cyclic or acyclic, halogenated, bisphosphorus, or evenpolymeric. Similarly, the azides can be alkyl, aryl, acyl or phosphoryl.In certain embodiments, such ligations are carried out under oxygen-freeanhydrous conditions. The proteins described herein can be modifiedusing the modified sugars described herein, including but not limited toUDP-GalNAz, using a Staudinger reaction (see, for example, Saxon, E.;Luchansky, S. J.; Hang, H. C.; Yu, C.; Lee, S. C.; Bertozzi, C. R.; J.Am. Chem. Soc.; 2002; 124(50); 14893-14902.).

In certain embodiments of the methods for labeling biomolecule utilizingStaudinger ligation described herein, the biomolecule can possess anazide moiety, whereupon the label possesses a phosphine moiety, whereasin other embodiments the biomolecule can possess a phosphine moiety, andthe label possesses an azide moiety.

Activated-Alkyne Chemistry

Azides and alkynes can undergo catalyst free [3+2] cycloaddition by ausing the reaction of activated alkynes with azides. Such catalyst free[3+2] cycloaddition can be used in methods described herein to conjugatea label to a biomolecule. Alkynes can be activated by ring strain suchas, by way of example only, eight membered ring structures, appendingelectron-withdrawing groups to such alkyne rings, or alkynes can beactivated by the addition of a Lewis acid such as, by way of exampleonly, Au(I) or Au(III).

In certain embodiments of the methods for labeling biomolecule utilizingactivated alkynes described herein, the biomolecule can possess an azidemoiety, whereupon the label possesses an activated alkyne moiety,whereas in other embodiments the biomolecule can possess an activatedalkyne moiety, and the label possesses an azide moiety.

Chemical modification of Post Translationally Modified Biomolecules

After biomolecules, including but not limited to proteins, have beenmodified either metabolically or enzymatically with azido moieties,alkyne moieties or phosphine moieties, they can be reacted underappropriate conditions to form conjugates with reporter molecules, solidsupports or carrier molecules. In certain embodiments, such proteinsused for such conjugations may be present as a cell lysate, as isolatedproteins, and/or as purified proteins, separated by gel electrophoresisor on a solid or semi-solid matrix. In certain embodiments, thebiomolecules are glycoproteins that have been modified, eithermetabolically or enzymatically, with azide-containing sugars,alkyne-containing sugars or phosphine-containing sugars.

In the methods and compositions described herein the azide moiety,alkyne moiety or phosphine moiety is used as a reactive functional groupon the modified biomolecule wherein an azide reactive moiety on areporter molecule, a solid support or a carrier molecule, or an alkynereactive moiety on a reporter molecule, a solid support or a carriermolecule, or a phosphine reactive moiety on a reporter molecule, a solidsupport or a carrier molecule is reacted with a modified biomolecule toform a covalent conjugate comprising the biomolecule and at least onereporter molecule, at least one solid support and/or at least onecarrier molecule. In certain embodiments such biomolecules are proteins,while in other embodiments such proteins are glycoproteins.

In certain embodiments of the methods and compositions described herein,a glycoprotein containing an azide moiety can be selectively labeledwith a reporter molecule, a solid support and/or a carrier molecule thatcontain azide reactive groups including, but not limited to, a terminalalkyne, an activated alkyne, or a phosphine. In other embodiments, aglycoprotein containing an alkyne moiety can be selectively labeled witha reporter molecule, a solid support and/or a carrier molecule thatcontain alkyne reactive groups including, but not limited to, an azidemoiety. In other embodiments, a glycoprotein containing an activatedalkyne moiety can be selectively labeled with a reporter molecule, asolid support and/or a carrier molecule that contain alkyne reactivegroups including, but not limited to, an azide moiety.

In certain embodiments, two azide-reactive groups are use to labelbiomolecules: the first is an alkyne moiety used in a “click” chemistryreaction, and the second is a phosphine, such as a triarylphosphine,used in a Staudinger ligation. In one embodiment, “click” chemistry isutilized to form a conjugate with a glycoprotein containing an azidemoiety and a reporter molecule, solid support or carrier molecule,wherein the reporter molecule, solid support and carrier moleculecontain an alkyne moiety. In another embodiment, “click” chemistry isutilized to form a conjugate with a glycoprotein containing an alkynemoiety and a reporter molecule, solid support and/or carrier molecule,wherein the reporter molecule, solid support and carrier moleculecontain an azide moiety. In another embodiment, a Staudinger ligation isutilized to form a conjugate with a glycoprotein containing an azidemoiety and a reporter molecule, solid support and/or carrier molecule,wherein the reporter molecule, solid support and carrier moleculecontain an triaryl phosphine moiety. In another embodiment, a Staudingerligation is utilized to form a conjugate with a glycoprotein containinga triaryl phosphine moiety and a reporter molecule, solid support and/orcarrier molecule, wherein the reporter molecule, solid support andcarrier molecule contain an azide moiety. The methods described hereinare not intended to be limited to these two azide-reactive groups, orchemical reactions, but it is envisioned that any chemical reactionutilizing an azide-reactive group attached to a reporter molecule, solidsupport or carrier molecule can be used with the azide modifiedglycoproteins described herein.

Protein can be modified using nucleophilic substitution reactions withamines, carboxylates or sulfhydryl groups which are found more commonlyon the surface of proteins. However, the methods described hereinutilize cycloaddition reactions, rather than nucleophilic substitutionreactions, for selective modifications of proteins. Thus proteinsdescribed herein can be modified, with the modified sugars describedherein, including but not limited to UDP-GalNAz, with extremely highselectivity. Such reactions can be carried out at room temperature inaqueous conditions with excellent regioselectivity by the addition ofcatalytic amounts of Cu(I) salts to the reaction mixture. See, e.g.,Tomoe, et al., (2002) Org. Chem. 67:3057-3064; and, Rostovtsev, et al.,(2002) Angew. Chem. Int. Ed. 41:2596-2599. The resulting five-memberedring resulting from “click” chemistry cycloaddition is not generallyreversible in reducing environments and is stable against hydrolysis forextended periods in aqueous environments. Thus, glycoproteins attachedto a labeling agent, a detection agent, a reporter molecule, a solidsupport or a carrier molecule via such five-membered ring are stable inreducing environments.

The reporter molecules, solid supports and carrier molecules used in themethods and compositions described herein, can contain at least onealkyne moiety or at least one phosphine moiety capable of reacting withan azide moiety. The reporter molecules, solid supports and carriermolecules used in the methods and compositions described herein, cancontain at least one azide moiety capable of reacting with an alkynemoiety or a phosphine moiety. The reporter molecules, solid supports andcarrier molecules used in the methods and compositions described herein,can contain at least one phosphine moiety capable of reacting with anazide moiety. In certain embodiments, the phosphine moieties of thereporter molecules solid supports and carrier molecules described hereinare triaryl phosphine moieties.

In certain embodiments, the reporter molecules used in the methods andcompositions described herein can include, but are not limited tolabels, while the solid supports can include, but are not limited to,solid support resins, microtiter plates and microarray slides. Thecarrier molecules can include, but are not limited to, affinity tags,nucleotides, oligonucleotides and polymers.

Reporter Molecules

The reporter molecules used in the methods and compositions providedherein include any directly or indirectly detectable reporter moleculeknown by one skilled in the art that can be covalently attached to amodified biomolecule, including a protein such as a glycoprotein. Suchmodified glycoproteins can be azide modified glycoproteins, alkynemodified glycoproteins or phosphine modified glycoproteins. In certainembodiments, the reporter molecules used in the methods and compositionsprovided herein include any directly or indirectly detectable reportermolecule known by one skilled in the art that can be covalently attachedto an azide modified glycoprotein, an alkyne modified glycoprotein or aphosphine modified glycoprotein.

Reporter molecules used in the methods and compositions described hereincan contain, but are not limited to, a chromophore, a fluorophore, afluorescent protein, a phosphorescent dye, a tandem dye, a particle, ahapten, an enzyme and a radioisotope. In certain embodiments, suchreporter molecules include fluorophores, fluorescent proteins, haptens,and enzymes.

A fluorophore used in a reporter molecule in the methods andcompositions described herein, can contain one or more aromatic orheteroaromatic rings, that are optionally substituted one or more timesby a variety of substituents, including without limitation, halogen,nitro, cyano, alkyl, perfluoroalkyl, alkoxy, alkenyl, alkynyl,cycloalkyl, arylalkyl, acyl, aryl or heteroaryl ring system, benzo, orother substituents typically present on fluorophores known in the art.

A fluorophore used in a reporter molecule in the methods andcompositions described herein, is any chemical moiety that exhibits anabsorption maximum at wavelengths greater than 280 nm, and retains itsspectral properties when covalently attached to a labeling reagent suchas, by way of example only, an azide, and alkyne or a phosphine.Fluorophores used as in reporter molecule in the methods andcompositions described herein include, without limitation; a pyrene(including any of the corresponding derivative compounds disclosed inU.S. Pat. No. 5,132,432), an anthracene, a naphthalene, an acridine, astilbene, an indole or benzoindole, an oxazole or benzoxazole, athiazole or benzothiazole, a 4-amino-7-nitrobenz-2-oxa-1,3-diazole(NBD), a cyanine (including any corresponding compounds in U.S. Ser.Nos. 09/968,401 and 09/969,853), a carbocyanine (including anycorresponding compounds in U.S. Ser. Nos. 09/557,275; 09/969,853 and09/968,401; U.S. Pat. Nos. 4,981,977; 5,268,486; 5,569,587; 5,569,766;5,486,616; 5,627,027; 5,808,044; 5,877,310; 6,002,003; 6,004,536;6,008,373; 6,043,025; 6,127,134; 6,130,094; 6,133,445; and publicationsWO 02/26891, WO 97/40104, WO 99/51702, WO 01/21624; EP 1 065 250 A1), acarbostyryl, a porphyrin, a salicylate, an anthranilate, an azulene, aperylene, a pyridine, a quinoline, a borapolyazaindacene (including anycorresponding compounds disclosed in U.S. Pat. Nos. 4,774,339;5,187,288; 5,248,782; 5,274,113; and 5,433,896), a xanthene (includingany corresponding compounds disclosed in U.S. Pat. Nos. 6,162,931;6,130,101; 6,229,055; 6,339,392; 5,451,343 and U.S. Ser. No.09/922,333), an oxazine (including any corresponding compounds disclosedin U.S. Pat. No. 4,714,763) or a benzoxazine, a carbazine (including anycorresponding compounds disclosed in U.S. Pat. No. 4,810,636), aphenalenone, a coumarin (including an corresponding compounds disclosedin U.S. Pat. Nos. 5,696,157; 5,459,276; 5,501,980 and 5,830,912), abenzofuran (including an corresponding compounds disclosed in U.S. Pat.Nos. 4,603,209 and 4,849,362) and benzphenalenone (including anycorresponding compounds disclosed in U.S. Pat. No. 4,812,409) andderivatives thereof. As used herein, oxazines include resorufins(including any corresponding compounds disclosed in U.S. Pat. No.5,242,805), aminooxazinones, diaminooxazines, and theirbenzo-substituted analogs.

Xanthene type fluorophores used in reporter molecule in the methods andcompositions described herein include, but are not limited to, afluorescein, a rhodol (including any corresponding compounds disclosedin U.S. Pat. Nos. 5,227,487 and 5,442,045), or a rhodamine (includingany corresponding compounds in U.S. Pat. Nos. 5,798,276; 5,846,737; U.S.Ser. No. 09/129,015). As used herein, fluorescein includes benzo- ordibenzofluoresceins, seminaphthofluoresceins, or naphthofluoresceins.Similarly, as used herein rhodol includes seminaphthorhodafluors(including any corresponding compounds disclosed in U.S. Pat. No.4,945,171). In certain embodiments, the fluorophore is a xanthene thatis bound via a linkage that is a single covalent bond at the 9-positionof the xanthene. In other embodiments, the xanthenes include derivativesof 3H-xanthen-6-ol-3-one attached at the 9-position, derivatives of6-amino-3H-xanthen-3-one attached at the 9-position, or derivatives of6-amino-3H-xanthen-3-imine attached at the 9-position.

In certain embodiments, the fluorophores used in reporter molecules inthe methods and compositions described herein include xanthene (rhodol,rhodamine, fluorescein and derivatives thereof) coumarin, cyanine,pyrene, oxazine and borapolyazaindacene. In other embodiments, suchfluorophores are sulfonated xanthenes, fluorinated xanthenes, sulfonatedcoumarins, fluorinated coumarins and sulfonated cyanines.

In other embodiments, the fluorophores used in reporter molecules in themethods and compositions described herein, wherein such fluorophoreshave been modified with azide moieties or alkyne moieties. When used in“click” chemistry reaction such fluorophores form triazole productswhich do not requires UV excitation and overcome any quenching effectdue to conjugation of azido or alkyne groups to the fluorescent-system.

The choice of the fluorophore attached to the labeling reagent willdetermine the absorption and fluorescence emission properties of thelabeling reagent, modified glycoprotein and immuno-labeled complex.Physical properties of a fluorophore label that can be used fordetection of modified glycoproteins and an immuno-labeled complexinclude, but are not limited to, spectral characteristics (absorption,emission and stokes shift), fluorescence intensity, lifetime,polarization and photo-bleaching rate, or combination thereof. All ofthese physical properties can be used to distinguish one fluorophorefrom another, and thereby allow for multiplexed analysis. In certainembodiments, the fluorophore has an absorption maximum at wavelengthsgreater than 480 nm. In other embodiments, the fluorophore absorbs at ornear 488 nm to 514 nm (particularly suitable for excitation by theoutput of the argon-ion laser excitation source) or near 546 nm(particularly suitable for excitation by a mercury arc lamp).

Many of fluorophores can also function as chromophores and thus thedescribed fluorophores are also chromophores used in reporter moleculesin the methods and compositions described herein.

In addition to fluorophores, enzymes also find use as labels for thedetection reagents/reporter molecules used in the methods andcompositions described herein. Enzymes are desirable labels becauseamplification of the detectable signal can be obtained resulting inincreased assay sensitivity. The enzyme itself does not produce adetectable response but functions to break down a substrate when it iscontacted by an appropriate substrate such that the converted substrateproduces a fluorescent, colorimetric or luminescent signal. Enzymesamplify the detectable signal because one enzyme on a labeling reagentcan result in multiple substrates being converted to a detectablesignal. This is advantageous where there is a low quantity of targetpresent in the sample or a fluorophore does not exist that will givecomparable or stronger signal than the enzyme. However, fluorophores aremost preferred because they do not require additional assay steps andthus reduce the overall time required to complete an assay. The enzymesubstrate is selected to yield the preferred measurable product, e.g.colorimetric, fluorescent or chemiluminescence. Such substrates areextensively used in the art, many of which are described in theMOLECULAR PROBES HANDBOOK, supra.

In certain embodiments, colorimetric or fluorogenic substrate and enzymecombination use oxidoreductases such as, by way of example only,horseradish peroxidase and a substrate such as, by way of example only,3,3′-diaminobenzidine (DAB) or 3-amino-9-ethylcarbazole (AEC), whichyield a distinguishing color (brown and red, respectively). Othercolorimetric oxidoreductase substrates used with the enzymatic reportermolecules described herein include, but are not limited to:2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS),o-phenylenediamine (OPD), 3,3′,5,5′-tetramethylbenzidine (TMB),o-dianisidine, 5-aminosalicylic acid, 4-chloro-1-naphthol. Fluorogenicsubstrates used with the enzymatic reporter molecules described hereininclude, but are not limited to, homovanillic acid or4-hydroxy-3-methoxyphenylacetic acid, reduced phenoxazines and reducedbenzothiazines, including Amplex® Red reagent and its variants (U.S.Pat. No. 4,384,042), Amplex UltraRed and its variants in (WO05042504)and reduced dihydroxanthenes, including dihydrofluoresceins (U.S. Pat.No. 6,162,931) and dihydrorhodamines including dihydrorhodamine 123.Peroxidase substrates can be used with the enzymatic reporter moleculesdescribed herein. Such peroxide substrates include, but are not limitedto, tyramides (U.S. Pat. Nos. 5,196,306; 5,583,001 and 5,731,158) whichrepresent a unique class of peroxidase substrates in that they can beintrinsically detectable before action of the enzyme but are “fixed inplace” by the action of a peroxidase in the process described astyramide signal amplification (TSA). These substrates are extensivelyutilized to label targets in samples that are cells, tissues or arraysfor their subsequent detection by microscopy, flow cytometry, opticalscanning and fluorometry.

In other embodiments the colorimetric (and in some cases fluorogenic)substrates and enzymes combination used in reporter molecules describedherein include a phosphatase enzyme such as, by way of example only, anacid phosphatase, an alkaline phosphatase or a recombinant version ofsuch a phosphatase. A colorimetric substrate used in combination withsuch phosphatases include, but are not limited to,5-bromo-6-chloro-3-indolyl phosphate (BCIP), 6-chloro-3-indolylphosphate, 5-bromo-6-chloro-3-indolyl phosphate, p-nitrophenylphosphate, or o-nitrophenyl phosphate or with a fluorogenic substratesuch as 4-methylumbelliferyl phosphate,6,8-difluoro-7-hydroxy-4-methylcoumarinyl phosphate (DiFMUP, U.S. Pat.No. 5,830,912), fluorescein diphosphate, 3-O-methylfluoresceinphosphate, resorufin phosphate,9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl)phosphate (DDAOphosphate), or ELF 97, ELF 39 or related phosphates (U.S. Pat. Nos.5,316,906 and 5,443,986).

Other enzymes used in reporter molecules described herein includeglycosidases, including, but not limited to, beta-galactosidase,beta-glucuronidase and beta-glucosidase. The colorimetric substratesused with such enzymes include, but are not limited to,5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-gal) and similarindolyl galactosides, glucosides, and glucuronides, o-nitrophenylbeta-D-galactopyranoside (ONPG) and p-nitrophenylbeta-D-galactopyranoside. Preferred fluorogenic substrates includeresorufin beta-D-galactopyranoside, fluorescein digalactoside (FDG),fluorescein diglucuronide and their structural variants (U.S. Pat. Nos.5,208,148; 5,242,805; 5,362,628; 5,576,424 and 5,773,236),4-methylumbelliferyl beta-D-galactopyranoside, carboxyumbelliferylbeta-D-galactopyranoside and fluorinated coumarinbeta-D-galactopyranosides (U.S. Pat. No. 5,830,912).

Additional enzymes used in reporter molecules described herein include,but are not limited to, hydrolases such as cholinesterases andpeptidases, oxidases such as glucose oxidase and cytochrome oxidases,and reductases for which suitable substrates are known.

Enzymes and their appropriate substrates that produce chemiluminescencecan also be used in reporter molecules described herein. Such enzymesinclude, but are not limited to, natural and recombinant forms ofluciferases and aequorins. In addition, the chemiluminescence-producingsubstrates for phosphatases, glycosidases and oxidases such as thosecontaining stable dioxetanes, luminol, isoluminol and acridinium estersan also be used in reporter molecules described herein.

In addition to enzymes, haptens can be used in label/reporter moleculesdescribed herein. In certain embodiments, such haptens include hormones,naturally occurring and synthetic drugs, pollutants, allergens, affectormolecules, growth factors, chemokines, cytokines, lymphokines, aminoacids, peptides, chemical intermediates, nucleotides, biotin and thelike. Biotin is useful because it can function in an enzyme system tofurther amplify the detectable signal, and it can function as a tag tobe used in affinity chromatography for isolation purposes. For detectionpurposes, an enzyme conjugate that has affinity for biotin is used, suchas, by way of example only, avidin-Horse Radish Peroxidase (HRP).Subsequently a peroxidase substrate as described herein can be added toproduce a detectable signal.

Fluorescent proteins can also be used in label/reporter moleculesdescribed herein for use in the methods, compositions and labelingreagents described herein. Non-limiting examples of such fluorescentproteins include green fluorescent protein (GFP) and thephycobiliproteins and the derivatives thereof. The fluorescent proteins,especially phycobiliprotein, are particularly useful for creating tandemdye labeled labeling reagents. These tandem dyes comprise a fluorescentprotein and a fluorophore for the purposes of obtaining a larger stokesshift wherein the emission spectra is farther shifted from thewavelength of the fluorescent protein's absorption spectra. This isparticularly advantageous for detecting a low quantity of a target in asample wherein the emitted fluorescent light is maximally optimized, inother words little to none of the emitted light is reabsorbed by thefluorescent protein. The fluorescent protein and fluorophore function asan energy transfer pair wherein the fluorescent protein emits at thewavelength that the fluorophore absorbs and the fluorophore then emitsat a wavelength farther from the fluorescent proteins emissionwavelength than could have been obtained with only the fluorescentprotein. A particularly useful combination is the phycobiliproteinsdisclosed in U.S. Pat. Nos. 4,520,110; 4,859,582; 5,055,556 and thesulforhodamine fluorophores disclosed in U.S. Pat. No. 5,798,276, or thesulfonated cyanine fluorophores disclosed in U.S. Ser. Nos. 09/968/401and 09/969/853; or the sulfonated xanthene derivatives disclosed in U.S.Pat. No. 6,130,101 and those combinations disclosed in U.S. Pat. No.4,542,104. Alternatively, the fluorophore functions as the energy donorand the fluorescent protein is the energy acceptor.

Carrier Molecules: Azide Reactive, Alkyne Reactive and PhosphineReactive

In the methods and compositions described herein the modifiedbiomolecules can be conjugated to a carrier molecule. In certainembodiments provided herein proteins, such as glycoproteins, arecovalently conjugated to a carrier molecule. This includes, but is notlimited to, any azide modified glycoprotein disclosed herein and anycarrier molecule disclosed herein. In certain embodiments, theglycoproteins contain at least one alkyne moiety or at least onephosphine moiety capable of reacting with a carrier molecule containingan azide moiety. In other embodiments, the glycoproteins contain atleast one azide moiety capable of reacting with a carrier moleculecontaining an alkyne moiety or a phosphine moiety. In other embodiments,the glycoproteins contain at least one phosphine moiety capable ofreacting with a carrier molecule containing an azide moiety. In certainembodiments, the phosphine moieties of the glycoproteins and carriermolecules are triaryl phosphine moieties.

A variety of carrier molecules can be used in the methods andcompositions described herein, including, but not limited to, antigens,steroids, vitamins, drugs, haptens, metabolites, toxins, environmentalpollutants, amino acids, peptides, proteins, nucleic acids, nucleic acidpolymers, carbohydrates, lipids, and polymers. In certain embodiments,the carrier molecule contain an amino acid, a peptide, a protein, apolysaccharide, a nucleoside, a nucleotide, an oligonucleotide, anucleic acid, a hapten, a psoralen, a drug, a hormone, a lipid, a lipidassembly, a synthetic polymer, a polymeric microparticle, a biologicalcell, a virus or combinations thereof.

In other embodiments, the carrier molecule is selected from a hapten, anucleotide, an oligonucleotide, a nucleic acid polymer, a protein, apeptide or a polysaccharide. In still other embodiments, the carriermolecule is an amino acid, a peptide, a protein, a polysaccharide, anucleoside, a nucleotide, an oligonucleotide, a nucleic acid, a hapten,a psoralen, a drug, a hormone, a lipid, a lipid assembly, a tyramine, asynthetic polymer, a polymeric microparticle, a biological cell,cellular components, an ion chelating moiety, an enzymatic substrate ora virus. In further embodiments, the carrier molecule is an antibody orfragment thereof, an antigen, an avidin or streptavidin, a biotin, adextran, an IgG binding protein, a fluorescent protein, agarose, and anon-biological microparticle.

In certain embodiments wherein the carrier molecule is an enzymaticsubstrate, the enzymatic substrate is selected from an amino acid, apeptide, a sugar, an alcohol, alkanoic acid, 4-guanidinobenzoic acid, anucleic acid, a lipid, sulfate, phosphate, —CH₂OCO-alkyl andcombinations thereof. In certain embodiments, such enzyme substrates canbe cleaved by enzymes selected from peptidases, phosphatases,glycosidases, dealkylases, esterases, guanidinobenzotases, sulfatases,lipases, peroxidases, histone deacetylases, exonucleases, reductases,endoglycoceramidases and endonucleases.

In other embodiments, the carrier molecule is an amino acid (includingthose that are protected or are substituted by phosphates,carbohydrates, or C₁ to C₂₂ carboxylic acids), or a polymer of aminoacids such as a peptide or protein. In a related embodiment, the carriermolecule contains at least five amino acids, more preferably 5 to 36amino acids. Such peptides include, but are not limited to,neuropeptides, cytokines, toxins, protease substrates, and proteinkinase substrates. Other peptides may function as organelle localizationpeptides, that is, peptides that serve to target the conjugated compoundfor localization within a particular cellular substructure by cellulartransport mechanisms, including, but not limited to, nuclearlocalization signal sequences. In certain embodiments, the proteincarrier molecules include enzymes, antibodies, lectins, glycoproteins,histones, albumins, lipoproteins, avidin, streptavidin, protein A,protein G, phycobiliproteins and other fluorescent proteins, hormones,toxins and growth factors. In other embodiments, the protein carriermolecule is an antibody, an antibody fragment, avidin, streptavidin, atoxin, a lectin, or a growth factor. In further embodiments, the carriermolecules contain haptens including, but not limited to, biotin,digoxigenin and fluorophores.

The carrier molecules used in the methods and composition describedherein can also contain a nucleic acid base, nucleoside, nucleotide or anucleic acid polymer, optionally containing an additional linker orspacer for attachment of a fluorophore or other ligand, such as analkynyl linkage (U.S. Pat. No. 5,047,519), an aminoallyl linkage (U.S.Pat. No. 4,711,955) or other linkage. In other embodiments, thenucleotide carrier molecule is a nucleoside or a deoxynucleoside or adideoxynucleoside, while in other embodiments, the carrier moleculecontains a peptide nucleic acid (PNA) sequence or a locked nucleic acid(LNA) sequence. In certain embodiments, the nucleic acid polymer carriermolecules are single- or multi-stranded, natural or synthetic DNA or RNAoligonucleotides, or DNA/RNA hybrids, or incorporating an unusual linkersuch as morpholine derivatized phosphates (AntiVirals, Inc., CorvallisOreg.), or peptide nucleic acids such as N-(2-aminoethyl)glycine units,where the nucleic acid contains fewer than 50 nucleotides, moretypically fewer than 25 nucleotides.

The carrier molecules used in the methods and composition describedherein can also contain a carbohydrate or polyol, including apolysaccharide, such as dextran, FICOLL, heparin, glycogen, amylopectin,mannan, inulin, starch, agarose and cellulose, or a polymer such as apoly(ethylene glycol). In certain embodiments, the polysaccharidecarrier molecule includes dextran, agarose or FICOLL.

The carrier molecules used in the methods and composition describedherein can also include a lipid including, but not limited to,glycolipids, phospholipids, and sphingolipids. In certain embodiments,such lipids contain 6-25 carbons. In other embodiments, the carriermolecules include a lipid vesicle, such as a liposome, or is alipoprotein (see below). Some lipophilic substituents are useful forfacilitating transport of the conjugated dye into cells or cellularorganelles. In certain embodiments, the carrier molecule that possess alipophilic substituent can be used to target lipid assemblies such asbiological membranes or liposomes by non-covalent incorporation of thedye compound within the membrane, e.g., for use as probes for membranestructure or for incorporation in liposomes, lipoproteins, films,plastics, lipophilic microspheres or similar materials.

The carrier molecules used in the methods and composition describedherein can also be a cell, cellular systems, cellular fragment, orsubcellular particles, including virus particles, bacterial particles,virus components, biological cells (such as animal cells, plant cells,bacteria, or yeast), or cellular components. Non-limiting examples ofsuch cellular components that are useful as carrier molecules in themethods and composition described herein include lysosomes, endosomes,cytoplasm, nuclei, histones, mitochondria, Golgi apparatus, endoplasmicreticulum and vacuoles.

The carrier molecules used in the methods and composition describedherein can also non-covalently associates with organic or inorganicmaterials.

The carrier molecules used in the methods and composition describedherein can also include a specific binding pair member wherein theglycoproteins described herein can be conjugated to a specific bindingpair member and used in the formation of a bound pair. In certainembodiments, the presence of a labeled specific binding pair memberindicates the location of the complementary member of that specificbinding pair; each specific binding pair member having an area on thesurface or in a cavity which specifically binds to, and is complementarywith, a particular spatial and polar organization of the other. Incertain embodiments, the dye compounds (fluorophores or chromophores)described herein function as a reporter molecule for the specificbinding pair. Exemplary binding pairs are set forth in Table 2.

TABLE 2 Representative Specific Binding Pairs antigen antibody biotinavidin (or streptavidin or anti-biotin) IgG* protein A or protein G drugdrug receptor folate folate binding protein toxin toxin receptorcarbohydrate lectin or carbohydrate receptor peptide peptide receptorprotein protein receptor enzyme substrate enzyme DNA (RNA) cDNA (cRNA)†hormone hormone receptor ion chelator *IgG is an immunoglobulin †cDNAand cRNA are the complementary strands used for hybridization

In a particular aspect the carrier molecule, used in the methods andcompositions described herein, is an antibody fragment, such as, but notlimited to, anti-Fc, an anti-Fc isotype, anti-J chain, anti-kappa lightchain, anti-lambda light chain, or a single-chain fragment variableprotein; or a non-antibody peptide or protein, such as, for example butnot limited to, soluble Fc receptor, protein G, protein A, protein L,lectins, or a fragment thereof. In one aspect the carrier molecule is aFab fragment specific to the Fc portion of the target-binding antibodyor to an isotype of the Fc portion of the target-binding antibody (U.S.Ser. No. 10/118,204). The monovalent Fab fragments are typicallyproduced from either murine monoclonal antibodies or polyclonalantibodies generated in a variety of animals, for example but notlimited to, rabbit or goat. These fragments can be generated from anyisotype such as murine IgM, IgG₁, IgG_(2a), IgG_(2b) or IgG₃.

In alternative embodiments, a non-antibody protein or peptide such asprotein G, or other suitable proteins, can be used alone or coupled withalbumin. Preferred albumins include human and bovine serum albumins orovalbumin. Protein A, G and L are defined to include those proteinsknown to one skilled in the art or derivatives thereof that comprise atleast one binding domain for IgG, i.e. proteins that have affinity forIgG. These proteins can be modified but do not need to be and areconjugated to a reactive moiety in the same manner as the other carriermolecules described.

In another aspect, the carrier molecules, used in the methods andcompositions described herein, can be whole intact antibodies. Antibodyis a term of the art denoting the soluble substance or molecule secretedor produced by an animal in response to an antigen, and which has theparticular property of combining specifically with the antigen thatinduced its formation. Antibodies themselves also serve are antigens orimmunogens because they are glycoproteins and therefore are used togenerate anti-species antibodies. Antibodies, also known asimmunoglobulins, are classified into five distinct classes—IgG, IgA,IgM, IgD, and IgE. The basic IgG immunoglobulin structure consists oftwo identical light polypeptide chains and two identical heavypolypeptide chains (linked together by disulfide bonds).

When IgG is treated with the enzyme papain a monovalent antigen-bindingfragment can be isolated, referred herein to as a Fab fragment. When IgGis treated with pepsin (another proteolytic enzyme), a larger fragmentis produced, F(ab′)₂. This fragment can be split in half by treatingwith a mild reducing buffer that results in the monovalent Fab′fragment. The Fab′ fragment is slightly larger than the Fab and containsone or more free sulfhydryls from the hinge region (which are not foundin the smaller Fab fragment). The term “antibody fragment” is usedherein to define the Fab′, F(ab′)₂ and Fab portions of the antibody. Itis well known in the art to treat antibody molecules with pepsin andpapain in order to produce antibody fragments (Gorevic et al., Methodsof Enzyol., 116:3 (1985)).

The monovalent Fab fragments used as carrier molecules in the methodsand compositions described herein are produced from either murinemonoclonal antibodies or polyclonal antibodies generated in a variety ofanimals that have been immunized with a foreign antibody or fragmentthereof (U.S. Pat. No. 4,196,265 discloses a method of producingmonoclonal antibodies). Typically, secondary antibodies are derived froma polyclonal antibody that has been produced in a rabbit or goat but anyanimal known to one skilled in the art to produce polyclonal antibodiescan be used to generate anti-species antibodies. The term “primaryantibody” describes an antibody that binds directly to the antigen asopposed to a “secondary antibody” that binds to a region of the primaryantibody. Monoclonal antibodies are equal, and in some cases, preferredover polyclonal antibodies provided that the ligand-binding antibody iscompatible with the monoclonal antibodies that are typically producedfrom murine hybridoma cell lines using methods well known to one skilledin the art.

In one aspect the antibodies used as carrier molecules in the methodsand compositions described herein are generated against only the Fcregion of a foreign antibody. Essentially, the animal is immunized withonly the Fc region fragment of a foreign antibody, such as murine. Thepolyclonal antibodies are collected from subsequent bleeds, digestedwith an enzyme, pepsin or papain, to produce monovalent fragments. Thefragments are then affinity purified on a column comprising wholeimmunoglobulin protein that the animal was immunized against or just theFc fragments.

Solid Supports: Azide Reactive, Alkyne Reactive or Phosphine Reactive

In an aspect of the methods and composition described herein, themodified biomolecules can be covalently conjugated to a solid support.In certain embodiments provided herein proteins, such as glycoproteins,are covalently conjugated to a solid support. This includes, but is notlimited to, any azide modified glycoprotein disclosed herein and anysolid support disclosed herein. In certain embodiments, theglycoproteins contain at least one alkyne moiety or at least onephosphine moiety capable of reacting with a solid support containing anazide moiety. In other embodiments, the glycoproteins contain at leastone azide moiety capable of reacting with a solid support containing analkyne moiety or a phosphine moiety. In other embodiments, theglycoproteins contain at least one phosphine moiety capable of reactingwith a solid support containing an azide moiety. In certain embodiments,the phosphine moieties of the glycoproteins and solid supports aretriaryl phosphine moieties.

A variety of solid supports can be used in the methods and compositionsdescribed herein. Such solid supports are not limited to a specific typeof support, and therefore a large number of supports are available andare known to one of ordinary skill in the art. Such solid supportsinclude, but are not limited to, solid and semi-solid matrixes, such asaerogels and hydrogels, resins, beads, biochips (including thin filmcoated biochips), microfluidic chip, a silicon chip, multi-well plates(also referred to as microtitre plates or microplates), membranes,conducting and nonconducting metals, glass (including microscope slides)and magnetic supports. Other non-limiting examples of solid supportsused in the methods and compositions described herein include silicagels, polymeric membranes, particles, derivatized plastic films,derivatized glass, derivatized silica, glass beads, cotton, plasticbeads, alumina gels, polysaccharides such as Sepharose, poly(acrylate),polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose,dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin,nitrocellulose, diazocellulose, polyvinylchloride, polypropylene,polyethylene (including poly(ethylene glycol)), nylon, latex bead,magnetic bead, paramagnetic bead, superparamagnetic bead, starch and thelike. In certain embodiments, the solid supports used in the methods andcompositions described herein are substantially insoluble in liquidphases.

In certain embodiments, the solid support may include a solid supportreactive functional group, including, but not limited to, hydroxyl,carboxyl, amino, thiol, aldehyde, halogen, nitro, cyano, amido, urea,carbonate, carbamate, isocyanate, sulfone, sulfonate, sulfonamide,sulfoxide, wherein such functional groups are used to covalently attachthe azide-containing glycoproteins described herein. In otherembodiments, the solid support may include a solid support reactivefunctional group, including, but not limited to, hydroxyl, carboxyl,amino, thiol, aldehyde, halogen, nitro, cyano, amido, urea, carbonate,carbamate, isocyanate, sulfone, sulfonate, sulfonamide, sulfoxide,wherein such functional groups are used to covalently attach thealkyne-containing glycoproteins described herein. In still otherembodiments, the solid support may include a solid support reactivefunctional group, including, but not limited to, hydroxyl, carboxyl,amino, thiol, aldehyde, halogen, nitro, cyano, amido, urea, carbonate,carbamate, isocyanate, sulfone, sulfonate, sulfonamide, sulfoxide,wherein such functional groups are used to covalently attach thephosphine-containing glycoproteins described herein. In otherembodiments, the solid supports include azide, alkyne or phosphinefunctional groups to covalently attach such modified glycoproteins.

A suitable solid phase support used in the methods and compositionsdescribed herein, can be selected on the basis of desired end use andsuitability for various synthetic protocols. By way of example only,where amide bond formation is desirable to attach the modifiedglycoproteins described herein to the solid support, resins generallyuseful in peptide synthesis may be employed, such as polystyrene (e.g.,PAM-resin obtained from Bachem Inc., Peninsula Laboratories, etc.),POLYHIPE™ resin (obtained from Aminotech, Canada), polyamide resin(obtained from Peninsula Laboratories), polystyrene resin grafted withpolyethylene glycol (TentaGel™, Rapp Polymere, Tubingen, Germany),polydimethyl-acrylamide resin (available from Milligen/Biosearch,California), or PEGA beads (obtained from Polymer Laboratories). Incertain embodiments, the modified glycoproteins described herein aredeposited onto a solid support in an array format. In certainembodiments, such deposition is accomplished by direct surface contactbetween the support surface and a delivery mechanism, such as a pin or acapillary, or by ink jet technologies which utilize piezoelectric andother forms of propulsion to transfer liquids from miniature nozzles tosolid surfaces. In the case of contact printing, robotic control systemsand multiplexed printheads allow automated microarray fabrication. Forcontactless deposition by piezoelectric propulsion technologies, roboticsystems also allow for automatic microarray fabrication using eithercontinuous and drop-on-demand devices.

Compositions

In one aspect, the modified biomolecules, reporter molecules and carriermolecules provided herein can be used to form a first composition thatincludes a modified biomolecule, a first reporter molecule, and acarrier molecule. In another embodiment, a second biomolecule thatincludes a first composition in combination with a second conjugate,wherein the second conjugate comprises a carrier molecule or solidsupport that is covalently bonded to a second reporter molecule. Thefirst and second reporter molecules have different structures andpreferably have different emission spectra. In other embodiments, thefirst and second reporter molecules are selected so that theirfluorescence emissions essentially do not overlap. In other embodiments,the reporter molecules have different excitation spectra, while in otherembodiments the reporter molecules have similar excitation wavelengthsand are excited by the same laser. In such compositions, the carriermolecule (or solid support) of the conjugates in the second compositionmay be the same or a different molecule. The discussion hereinpertaining to the identity of various carrier molecules is generallyapplicable to this embodiment as well as other embodiments.

In certain embodiments, modified proteins, such as glycoproteins,reporter molecules and carrier molecules provided herein can be used toform a first composition that includes a modified glycoprotein, a firstreporter molecule, and a carrier molecule. In another embodiment, asecond glycoprotein that includes a first composition in combinationwith a second conjugate, wherein the second conjugate comprises acarrier molecule or solid support that is covalently bonded to a secondreporter molecule. The first and second reporter molecules havedifferent structures and preferably have different emission spectra. Inother embodiments, the first and second reporter molecules are selectedso that their fluorescence emissions essentially do not overlap. Inother embodiments, the reporter molecules have different excitationspectra, while in other embodiments the reporter molecules have similarexcitation wavelengths and are excited by the same laser. In suchcompositions, the carrier molecule (or solid support) of the conjugatesin the second composition may be the same or a different molecule. Thediscussion herein pertaining to the identity of various carriermolecules is generally applicable to this embodiment as well as otherembodiments.

In another aspect, the modified biomolecules, reporter molecules andsolid supports provided herein can be used to form a first compositionthat comprises a modified biomolecule, a first reporter molecule, and asolid support. In another embodiment, a second composition that includesa first composition in combination with a second conjugate. The secondconjugate comprises a solid support or carrier molecule (describedherein) that is covalently bonded to a second reporter molecule. Thefirst and second reporter molecules have different structures andpreferably have different emission spectra. In other embodiments, thefirst and second reporter molecules are selected so that theirfluorescence emissions essentially do not overlap. In other embodiments,the reporter molecules have different excitation spectra, while in otherembodiments the reporter molecules have similar excitation wavelengthsand are excited by the same laser. In such composition, the solidsupport (or carrier molecule) of the conjugates in the secondcomposition may be the same or a different molecule. The discussionherein pertaining to the identity of various solid supports is generallyapplicable to this embodiment of the invention as well as otherembodiments.

In another aspect, the modified proteins, such as glycoproteins,reporter molecules and solid supports provided herein can be used toform a first composition that comprises a modified glycoprotein, a firstreporter molecule, and a solid support. In another embodiment, a secondcomposition that includes a first composition in combination with asecond conjugate. The second conjugate comprises a solid support orcarrier molecule (described herein) that is covalently bonded to asecond reporter molecule. The first and second reporter molecules havedifferent structures and preferably have different emission spectra. Inother embodiments, the first and second reporter molecules are selectedso that their fluorescence emissions essentially do not overlap. Inother embodiments, the reporter molecules have different excitationspectra, while in other embodiments the reporter molecules have similarexcitation wavelengths and are excited by the same laser. In suchcomposition, the solid support (or carrier molecule) of the conjugatesin the second composition may be the same or a different molecule. Thediscussion herein pertaining to the identity of various solid supportsis generally applicable to this embodiment of the invention as well asother embodiments.

Methods for Labeling Modified Biomolecules in Solution

Methods for forming modified biomolecule-reporter molecule conjugatesare described herein. In certain embodiments the biomolecules areglycoproteins that have been azido modified metabolically, enzymaticallyor chemically using the methods described herein. In one aspect themodified biomolecule-reporter molecule conjugates are formed in solutionand then separated using methods known in the art. In certainembodiments, the biomolecule labeled with the reporter molecule is amodified glycoprotein. In other embodiments, the biomolecule labeledwith the reporter molecule is an azido modified glycoprotein, alkynemodified glycoprotein, or phosphine modified glycoprotein.

Described herein are novel methods for forming conjugates in solutionwith azido modified biomolecules and a reporter molecule comprising aterminal alkyne under “click” chemistry conditions. In otherembodiments, “click” chemistry is used to form conjugates with alkynemodified biomolecules and a reporter molecule comprising an azide. Inother embodiments, Staudinger ligation is used to form conjugates withazide modified biomolecules and a reporter molecule comprising aphosphine, while other embodiments use Staudinger ligation to formconjugates with phosphine modified biomolecules and a reporter moleculecomprising an azide. Still other embodiments use activated alkynemodified biomolecules to form conjugates with reporter moleculescomprising azides, or azide modified biomolecules forming conjugateswith activated alkyne containing reporter molecules.

It was unexpectedly found that by adding a copper chelator to the“click” chemistry conjugation reaction the labeling efficiency andresolution after gel electrophoresis improved as compared to thosereactions without the addition of a copper chelator. In certainembodiments, the methods of labeling biomolecules using “click”chemistry, involve a biomolecule that includes an azido group and alabel that includes a terminal alkyne that are reacted in a mixture thatincludes copper (II), a reducing agent, and at least one copper (I)chelator, thereby producing a labeled biomolecule. In certainembodiments, the biomolecules labeled in such methods can bepolysaccharides, nucleic acids, proteins, or peptides. In certainembodiments, the biomolecules used in the labeling methods describedherein are glycoproteins

In certain embodiments, the biomolecules used in the labeling methodsdescribed herein are proteins, such as but not limited to glycoproteins.The labeling methods used to label proteins, including glycoproteins,include “click” chemistry or Staudinger ligation. In certainembodiments, the labeling of glycoproteins occurs by “click” chemistryin which a glycoprotein that includes an azido group and a label thatcomprises a terminal alkyne react in a mixture that includes copper(II), a reducing agent, and at least one copper chelator to produce alabeled glycoprotein. In certain embodiments, the labeling ofglycoproteins occurs by “click” chemistry in which a glycoprotein thatincludes an alkyne group and a label that comprises an azide react in amixture that includes copper (II), a reducing agent, and at least onecopper chelator to produce a labeled glycoprotein.

In other aspects provided herein, the methods of labeling biomoleculesusing “click” chemistry, wherein a biomolecule that includes an azidogroup and a label that comprises a terminal alkyne are reacted in amixture that includes copper (II), a reducing agent, and at least onecopper (I) chelator to produce a labeled biomolecule, results in thepreservation of the structural integrity of the labeled biomolecule. Inother embodiments, the biomolecules labeled in such cycloadditionreactions can be polysaccharides, nucleic acids, proteins, or peptides.In certain embodiments, the biomolecules used in such methods areproteins, such as but not limited to glycoproteins. In such methods aglycoprotein that includes an azido group and a label that comprises aterminal alkyne are reacted in a mixture that includes copper (II), areducing agent, and at least one copper (I) chelator to produce alabeled glycoprotein, and results in the preservation of the structuralintegrity of the labeled glycoprotein, wherein the structural integrityof the glycoprotein after labeling is not reduced. As described herein,the glycoproteins can be derivatized, for example, by in vivoincorporation of azido sugars or by in vitro enzymatic addition of azidosugars. In other embodiments, methods of labeling glycoproteins whereinthe structural integrity of the glycoprotein after labeling is notreduced includes “click” chemistry in which a glycoprotein that includesa terminal alkyne and a label that comprises an azido group are reactedin a mixture that includes copper (II), a reducing agent, and at leastone copper chelator to produce a labeled glycoprotein.

The methods for labeling biomolecules that comprise an azido group using“click” chemistry described herein can also be used for biomoleculesthat comprise a terminal alkyne, wherein the label to be reacted withthe biomolecule comprises an azido group. The methods for labeling anddetecting biomolecules that comprise an azido group using “click”chemistry described herein can also be used for biomolecules thatcomprise a terminal alkyne, wherein the label to be reacted with thebiomolecule comprises an azido group. In one embodiment, is a methodusing the “click” chemistry reaction described herein to formbiomolecule-reporter molecule conjugates in which the reaction mixtureincludes a reporter molecule with an azide moiety, an alkyne modifiedbiomolecule, copper (II) ions, at least one reducing agent and a copperchelator. In certain embodiments, such alkyne modified biomolecule arealkyne modified glycoproteins and such reporter molecule with an azidemoiety are any reporter molecule described herein. In other embodiments,such alkyne modified biomolecule are alkyne modified glycoproteins andsuch reporter molecule with an azide moiety are any fluorophore basedreporter molecule described herein.

Other methods provided herein, are methods for labeling and detectingseparated proteins, including but not limited to glycoproteins, usingthe “click” chemistry cycloaddition reaction described herein. Themethod includes: combining in a reaction mixture a biomolecule thatcomprises an azido group, a label that includes a terminal alkyne group,copper (II), a reducing agent, and a copper chelator; incubating thereaction mixture under conditions that promote chemical conjugation ofthe label to the biomolecule, separating the biomolecule using one ormore biochemical or biophysical separation techniques, and detecting thebiomolecule. In other embodiments, the method includes: combining in areaction mixture a biomolecule that comprises an alkyne group, a labelthat includes an azide group, copper (II), a reducing agent, and acopper chelator; incubating the reaction mixture under conditions thatpromote chemical conjugation of the label to the biomolecule, separatingthe biomolecule using one or more biochemical or biophysical separationtechniques, and detecting the biomolecule.

In another embodiment is a method for detecting modified biomolecules,wherein the method includes the steps of:

-   -   a) forming an azide-alkyne cycloaddition reaction mixture that        includes a reporter molecule having a terminal alkyne moiety, an        azido modified biomolecule, copper(II) ions, at least one        reducing agent and a copper chelator;    -   b) incubating the azide-alkyne cycloaddition reaction mixture        for a sufficient amount of time to form a biomolecule-reporter        molecule conjugate;    -   c) separating the biomolecule-reporter molecule conjugate by        size and/or weight of the biomolecule-reporter molecule        conjugate to form a separated biomolecule-reporter molecule        conjugate;    -   d) illuminating the separated biomolecule-reporter molecule        conjugate with an appropriate wavelength to form an illuminated        biomolecule-reporter molecule conjugate, and    -   e) observing the illuminated biomolecule-reporter molecule        conjugate wherein the biomolecules is detected.

In another embodiment is a method for detecting modified glycoproteins,wherein the method includes the steps of:

-   -   a) forming an azide-alkyne cycloaddition reaction mixture that        includes a reporter molecule having a terminal alkyne moiety, an        azido modified glycoprotein, copper(II) ions, at least one        reducing agent and a copper chelator;    -   b) incubating the azide-alkyne cycloaddition reaction mixture        for a sufficient amount of time to form a glycoprotein-reporter        molecule conjugate;    -   c) separating the glycoprotein-reporter molecule conjugate by        size and/or weight of the glycoprotein-reporter molecule        conjugate to form a separated glycoprotein-reporter molecule        conjugate;    -   d) illuminating the separated glycoprotein-reporter molecule        conjugate with an appropriate wavelength to form an illuminated        glycoprotein-reporter molecule conjugate, and    -   e) observing the illuminated glycoprotein-reporter molecule        conjugate wherein the glycoprotein is detected.

In another embodiment is a method for detecting modified biomolecules,wherein the method includes the steps of:

-   -   a) forming an azide-alkyne cycloaddition reaction mixture that        includes a reporter molecule having an azide moiety, an alkyne        modified biomolecule, copper(II) ions, at least one reducing        agent and a copper chelator;    -   b) incubating the azide-alkyne cycloaddition reaction mixture        for a sufficient amount of time to form a biomolecule-reporter        molecule conjugate;    -   c) separating the biomolecule-reporter molecule conjugate by        size and/or weight of the biomolecule-reporter molecule        conjugate to form a separated biomolecule-reporter molecule        conjugate;    -   d) illuminating the separated biomolecule-reporter molecule        conjugate with an appropriate wavelength to form an illuminated        biomolecule-reporter molecule conjugate, and    -   e) observing the illuminated biomolecule-reporter molecule        conjugate wherein the biomolecule is detected.

In another embodiment is a method for detecting modified glycoproteins,wherein the method includes the steps of:

-   -   a) forming an azide-alkyne cycloaddition reaction mixture that        includes a reporter molecule having an azide moiety, an alkyne        modified glycoprotein, copper(II) ions, at least one reducing        agent and a copper chelator;    -   b) incubating the azide-alkyne cycloaddition reaction mixture        for a sufficient amount of time to form a glycoprotein-reporter        molecule conjugate;    -   c) separating the glycoprotein-reporter molecule conjugate by        size and/or weight of the glycoprotein-reporter molecule        conjugate to form a separated glycoprotein-reporter molecule        conjugate;    -   d) illuminating the separated glycoprotein-reporter molecule        conjugate with an appropriate wavelength to form an illuminated        glycoprotein-reporter molecule conjugate, and    -   e) observing the illuminated glycoprotein-reporter molecule        conjugate wherein the glycoprotein is detected.

In addition such “click” chemistry reaction mixtures can include,without limitation, one or more buffers, polymers, salts, detergents, orsolubilizing agents. The reaction can be performed under anaerobicconditions, such as under nitrogen or argon gas, and can be performedfor any feasible length of time, such as, for example, from ten minutesto six hours, from about twenty minutes to about three hours, or fromabout thirty minutes to about two hours. The reaction can be performedat a wide range of temperatures, for example ranging from about 4degrees Celsius to about 50 degrees Celsius, and is preferably performedat temperatures between about 10 degrees and about 40 degrees, andtypically between about 15 degrees and about 30 degrees.

Separation and Detection

Another aspect provided herein are methods directed toward detectingmodified biomolecules after the modified biomolecules have been labeled,using “click” chemistry reactions, Saudinger ligation or activatedalkyne reactions, and separated using, for example, chromatographicmethods or electrophoresis methods such as, but not limited to, gelelectrophoresis. The modified biomolecules that can be labeled,separated and detected using the methods described herein include, butare not limited to, polysaccharides, nucleic acids, proteins, orpeptides and glycoproteins. In certain embodiments such biomoleculeshave been modified using the methods described herein. The separationmethods used to separate such modified biomolecules includes, but arenot limited to, thin layer or column chromatography (including, forexample, size exclusion, ion exchange, or affinity chromatography) orisoelectric focusing, gel electrophoresis, capillary electrophoresis,capillary gel electrophoresis, and slab gel electrophoresis. Gelelectrophoresis can be denaturing or nondenaturing gel electrophoresis,and can include denaturing gel electrophoresis followed by nondenaturinggel electrophoresis (e.g., “2D” gels). In certain embodiments, themodified biomolecules are used to form conjugates with a reportermolecule, a carrier molecule and/or a solid support prior to separationusing the methods described herein. In other embodiments, the modifiedbiomolecules are used to form conjugates with a reporter molecule, acarrier molecule and/or a solid support after separation using themethods described herein.

In other embodiments, the separation methods used in such separation anddetection methods can be any separation methods used for biomolecules,such as, for example, chromatography, capture to solid supports, andelectrophoresis. In certain embodiments, gel electrophoresis is used toseparate biomolecules, such as but not limited to proteins. Gelelectrophoresis is well known in the art, and in the context of thepresent invention can be denaturing or nondenaturing gel electrophoresisand can be 1D or 2D gel electrophoresis. FIG. 3 shows detection, via 2-Dgel electrophoresis, of proteins modified with Ac₄GlcNAz and labeledwith a fluorescent alkyne probe using click chemistry.

In certain embodiments of such separation and detection methods, gelelectrophoresis is used to separate proteins, including glycoproteins,and the separated proteins are detected in the gel by the attachedlabels. By way of example only, glycoproteins that have incorporatedazido sugars can be labeled in a solution reaction with a terminalalkyne-containing fluorophore, and the proteins can be optionallyfurther purified from the reaction mixture and electrophoresed on a 1Dor 2D gel. The proteins can be visualized in the gel using light of theappropriate wavelength to stimulate the fluorophore label.

Gel electrophoresis can use any feasible buffer system described hereinincluding, but not limited to, Tris-acetate, Tris-borate, Tris-glycine,BisTris and Bistris-Tricine. In certain embodiments, the electrophoresisgel used in the methods described herein comprise acrylamide, includingby way for example only, acrylamide at a concentration from about 2.5%to about 30%, or from about 5% to about 20%. In certain embodiments,such polyacrylamide electrophoresis gel comprise 1% to 10% crosslinker,including but not limited to, bisacrylamide. In certain embodiments, theelectrophoresis gel used in the methods described herein comprisesagarose, including by way for example only, agarose at concentrationfrom about 0.1% to about 5%, or from about 0.5% to about 4%, or fromabout 1% to about 3%. In certain embodiments, the electrophoresis gelused in the methods described herein comprises acrylamide and agarose,including by way for example only, electrophoresis gels comprising fromabout 2.5% to about 30% acrylamide and from about 0.1% to about 5%agarose, or from about 5% to about 20% acrylamide and from about 0.2% toabout 2.5% agarose. In certain embodiments, such polyacrylamide/agaroseelectrophoresis gel comprise 1% to 10% crosslinker, including but notlimited to, bisacrylamide. In certain embodiments, the gels used toseparate biomolecules can be gradient gels.

The methods described herein can be used to detect modified biomoleculesfor “in-gel” detection using slab gel electrophoresis or capillary gelelectrophoresis. In certain embodiments such modified biomolecules areglycoproteins. In one aspect, the method includes combining an azidomodified biomolecule, a label that includes a terminal alkyne, copper(II), a reducing agent, and a copper (I) chelator in a reaction mixture;incubating the reaction mixture under conditions that promote chemicalconjugation of the label to the biomolecule; separating the biomoleculeusing one or more biochemical separation techniques; and detecting thebiomolecule. The label used in such methods can be any label describedherein. The copper (I) chelator used in such methods can be any chelatordescribed herein. In certain embodiments, the copper (I) chelator use insuch methods is a 1,10 phenanthroline-containing copper (I) chelator. Inother embodiments, the copper(I) chelator is bathocuproine disulfonicacid (BCS; 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline disulfonate. Inother embodiments, the copper (I) chelator used in such methods can beused to chelate copper(II).

Without limitation to any specific mechanism, it is known that coppercan promote the cleavage of biomolecules such as proteins and nucleicacids. The addition of a copper chelator in such methods reduces thedetrimental effects of copper used in the “click” chemistry reactions,and thereby preserves the structural integrity of the biomolecules.Thus, the methods described herein preserve the structural integrity oflabeled and detected biomolecules, and thereby provide improved methodsof separating and detecting biomolecules labeled using “click”chemistry. In addition, the methods of detecting separated biomoleculesusing click chemistry, in which the structural integrity of theseparated molecules is preserved, improves the detection of suchbiomolecules.

In another embodiment of “in-gel” detection, the method includescombining an alkyne modified biomolecule that comprises a terminalalkyne, a label that includes an azido group, copper (II), a reducingagent, and a copper (I) chelator in a reaction mixture; incubating thereaction mixture under conditions that promote chemical conjugation ofthe label to the biomolecule; separating the labeled biomolecule usingone or more biochemical separation techniques; and detecting thebiomolecule. In these methods, the structural integrity of labeled anddetected biomolecules is preserved. The label used in such methods canbe any label described herein. The copper (I) chelator used in suchmethods can be any chelator described herein. In certain embodiments,the copper (I) chelator use in such methods is a 1,10phenanthroline-containing copper (I) chelator. In other embodiments, thecopper(I) chelator is bathocuproine disulfonic acid (BCS;2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline disulfonate. In otherembodiments, the copper (I) chelator used in such methods can be used tochelate copper(II).

In-gel fluorescence detection allows for quantitative differentialanalysis of protein glycosylation between different biological samplesand is amenable to multiplexing with other protein gel stains. Incertain embodiments of the methods described herein, utilizingfluorescent- and/or UV-excitable alkyne containing probes, orfluorescent- and/or UV-excitable azide containing probes, allow for themultiplexed detection of glycoproteins, phosphoproteins, and totalproteins in the same 1-D or 2-D gels. FIG. 4 shows “in gel” detection of40 and 50 kD azide-labeled model proteins, which were first labeled witha fluorescent alkyne tag and then separated on the gel.

In certain embodiments, the labels used in such separation and detectionmethods are any fluorophores described herein which has been derivatizedto contain an alkyne, an azide or a phosphine. In certain embodiments,such fluorophores include, but are not limited to, fluorescein,rhodamine, TAMRA, an Alexa dye, a SYPRO dye, or a BODIPY dye.

The method described herein can be used for multiplexed detection ofbiomolecules, such as proteins by labeling the proteins with labels ofdifferent specificities. For example, a total proteins stain, such asSYPRO Ruby can be used to stain a gel that includes proteins labeledusing a fluorophore with distinct spectral emission using the methods ofthe present invention. Proteins having other characteristics, such asoxidized proteins or phosphorylated proteins, can be detected in thesame gel by use of phosphoprotein specific labels used to stain the gel.FIG. 9 shows multiplexed Western blot detection of O-GlcNAc and Cofilin.

In another aspect, proteins, such as glycoproteins) can be labeled withan azido tag, electrophoresed on gels, and the resulting gels can beincubated with an alkyne tag, such as a fluorescent alkyne tag in thepresence of copper (I). Copper (I) can be added in its natural form(e.g. CuBr) or can be produced in situ from copper (II) compounds withthe addition of a reducing agent. The reducing agent used in suchmethods can be any reducing agent described herein, including but notlimited to, ascorbate or TCEP. Addition of a chelator that stabilizescopper (I) can enhance the chemical ligation. The fluorescent label usedin such methods can be any fluorophore described herein. The copper (I)chelator used in such methods can be any chelator described herein. Incertain embodiments, the copper (I) chelator use in such methods is a1,10 phenanthroline-containing copper (I) chelator. In otherembodiments, the copper(I) chelator is bathocuproine disulfonic acid(BCS; 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline disulfonate. Inother embodiments, the copper (I) chelator used in such methods can beused to chelate copper(II). After the ligation step, the gel is washedand the tagged proteins are visualized using standard fluorescencescanning devices.

In other embodiments, proteins, such as glycoproteins, can be labeledwith an alkyne tag, electrophoresed on gels, and the resulting gels canbe incubated with an azide tag, such as a fluorescent azide tag in thepresence of copper (I). Copper (I) can be added in its natural form(e.g. CuBr) or can be produced in situ from copper (II) compounds withthe addition of a reducing agent. The reducing agent used in suchmethods can be any reducing agent described herein, including but notlimited to, ascorbate or TCEP. Addition of a chelator that stabilizescopper (I) can enhance the chemical ligation. The fluorescent label usedin such methods can be any fluorophore described herein. The copper (I)chelator used in such methods can be any chelator described herein. Incertain embodiments, the copper (I) chelator use in such methods is a1,10 phenanthroline-containing copper (I) chelator. In otherembodiments, the copper(I) chelator is bathocuproine disulfonic acid(BCS; 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline disulfonate. Inother embodiments, the copper (I) chelator used in such methods can beused to chelate copper(II). After the ligation step, the gel is washedand the tagged proteins are visualized using standard fluorescencescanning devices.

In further embodiments, proteins, such as glycoproteins, can be labeledwith an azide tag, electrophoresed on gels, and the resulting gels canbe incubated with a phosphine tag, such as a fluorescent phosphinecontaining tag, using Staudinger ligation. After the ligation step, thegel is washed and the tagged proteins are visualized using standardfluorescence scanning devices. In such methods the use of copper, whichcontributes to the degradation of biomolecules such as proteins, can beavoided.

In another aspect, detection of proteins labeled using the methodsdescribed herein can be by Western blot, in which biomoleculesderivatized to include an azido group are labeled with a detectablelabel prior to gel electrophoresis and transferred to a blottingmembrane. Azido-containing biomolecules can be labeled, for example,with alkyl-biotin, and after electrophoretic separation and transfer toa blotting membrane, can be detected using streptavidin linked to anenzyme that converts a chromogenic substrate. Those skilled in the artwill appreciate that any feasible label that is directly detectable orindirectly detectable and can be derivatized to include a terminalalkyne or an azido group can be attached to a biomolecule that includesan azido group or a terminal alkyne and used to detect separatedbiomolecules, including separated biomolecules transferred to amembrane.

In other embodiments Western blotting analyses reveal glycoproteinsubclass detection sensitivities in the low femtomole range and allowfor multiplexing with protein-specific antibodies. In certainembodiments, biotin-alkyne probes, or biotin-azide probes, allow formultiplexed Western blot detection of glycoproteins and targetedproteins of interest using monoclonal or polyclonal antibodies. Theresults achieved with the combined glycoprotein detection strategydescribed herein, provide selectivity and sensitivity that is currentlyunachievable with commonly used lectin-based and antibody-basedglycoprotein detection technologies.

The methods described herein utilizing copper catalyzed cycloadditionchemistry can result in highly sensitive detection of proteins modifiedwith azides or alkynes, as shown by 1-D and 2-D fluorescent gelsensitivities on gel electrophoresis and Western blots (see FIGS. 8-10).In certain embodiments the detection sensitivity is in the low picomolerange, while in other embodiments the detection sensitivity is in themid-to-low femtomole range (see FIG. 6B), and the labeling efficiency

In certain embodiments, a label attached to a biomolecule, such as aprotein, using a “click” chemistry reaction with a copper (I) chelatoras disclosed herein, can also be used for the separation ofbiomolecules. By way of example only, affinity chromatography or beadcapture techniques can be used to separate biomolecules labeled withbiotin or other affinity tags using the methods described herein. Thecaptured molecules can be detected using the affinity tags or by othermeans, and/or further analyzed for structure or function.

Another aspect of “in gel” detection is the total detection of proteinsin electrophoresis gels or Western blot membranes using a “universalclick” chemistry in which phenylboronic acid-containing molecules aretethered via a linker to an azide moiety or an alkyne moiety. Thephenylboronic acid associates with the cis-diol moieties onglycoproteins which is stable, except under acidic conditions. Suchlabels can be used to modify glycoproteins after electrophoreticseparation with either azide or alkyne moieties which can then be usedto add a label via “click” chemistry, Staudinger ligation or activatedalkyne chemistry. The gel is then visualized to detect the labeledglycoproteins. In certain embodiments, glycoproteins of interest can beisolated by excising bands of interest after such labeling and treatingthe gel pieces under acidic conditions to reverse the association of thephenylboronic acid with the cis-diol moieties on glycoproteins, therebyreleasing the glycoproteins. The released glycoproteins can then beidentified using mass spectrometry.

Methods for Labeling Immobilized Modified Biomolecules

Another aspect provides a method for labeling modified biomolecules thathave been immobilized on a solid support. Solid supports used in suchmethods have been described herein, and can be solid or semi-solidmatrix. Such solid supports include, but are not limited to, glass,slides, arrays, silica particles, polymeric particles, microtiter platesand polymeric gels. In this aspect the biomolecules are modified usingthe methods described herein. In certain aspects it is advantageous tofirst immobilize the modified biomolecules and then to subsequently forma biomolecule conjugate comprising the biomolecule and a reportermolecule, carrier molecule and the solid support, wherein the reportermolecule, carrier molecule or solid support comprise a reactive groupused to form the conjugate. In certain embodiments such reactive groupsare alkynes for reacting with azides. In certain embodiments suchreactive groups are activated alkynes for reacting with azides. Incertain embodiments such reactive groups are phosphines for reactingwith azides. In certain embodiments such reactive groups are azides forreacting with alkynes. In certain embodiments, the conjugate is formedunder “click” chemistry conditions wherein the reporter molecule,carrier molecule or solid support comprises an alkyne or an azide. Inanother aspect the conjugate is formed under Staudinger ligationconditions wherein the reporter molecule, carrier molecule or solidsupport comprises a triaryl phosphine or an azide. In another aspect theconjugate is formed using activated alkynes wherein the reportermolecule, carrier molecule or solid support comprises an activatedalkyne or an azide.

In this aspect the biomolecules are metabolically, enzymatically orchemically modified with an azide containing moiety, an alkynecontaining moiety or a phosphine containing moiety using the methodsdescribed herein. In certain embodiments the modified biomolecule is anazido modified biomolecule, an alkyne modified biomolecule or aphosphine modified biomolecule. In certain embodiments, the azidomodified biomolecule is an azido modified glycoprotein. In certainembodiments, the alkyne modified biomolecule is an alkyne modifiedglycoprotein. In certain embodiments, the phosphine modified biomoleculeis a phosphine modified glycoprotein.

In certain embodiments, it is advantageous to first immobilize the azidomodified biomolecules and then to subsequently form the biomoleculeconjugate comprising a reporter molecule, carrier molecule or solidsupport wherein the reporter molecule, carrier molecule or solid supportcomprise an azide reactive group prior to forming the conjugate. Incertain embodiments, the conjugate is formed under “click” chemistryconditions wherein the reporter molecule, carrier molecule or solidsupport comprises a terminal alkyne. In another aspect the conjugate isformed under Staudinger ligation conditions wherein the reportermolecule, carrier molecule or solid support comprises a triarylphosphine. In certain embodiments, azido modified glycoproteins arefirst immobilized onto a solid support, and then subsequently used toform a modified glycoprotein conjugate comprising a reporter molecule,carrier molecule or solid support wherein the reporter molecule, carriermolecule or solid support comprise an azide reactive group prior toforming the conjugate. In certain embodiments, the conjugate is formedunder “click” chemistry conditions wherein the reporter molecule,carrier molecule or solid support comprises a terminal alkyne. Inanother aspect the conjugate is formed under Staudinger ligationconditions wherein the reporter molecule, carrier molecule or solidsupport comprises a triaryl phosphine.

In another aspect, the modified biomolecule is attached to a solidsupport using functional groups other than functional groups used in“click” chemistry or Staudinger ligation, whereupon the attachedmodified biomolecule is used to form a conjugate under “click” chemistryconditions or Staudinger ligation with reporter molecules, carriermolecule or another solid support that have functional groups used in“click” chemistry or Staudinger ligation. By way of example only, themodified biomolecule can be immobilized to a solid support usinghydroxyl, carboxyl, amino, thiol, aldehyde, halogen, nitro, cyano,amido, urea, carbonate, carbamate, isocyanate, sulfone, sulfonate,sulfonamide or sulfoxide functional groups.

In this aspect the biomolecules are modified with an azide containingmoiety, an alkyne containing moiety or a phosphine containing moietyusing the methods described herein. In certain embodiments the modifiedbiomolecule is an azido modified biomolecule, an alkyne modifiedbiomolecule or a phosphine modified biomolecule. In certain embodiments,the azido modified biomolecule is an azido modified glycoprotein. Incertain embodiments, the alkyne modified biomolecule is an alkynemodified glycoprotein. In certain embodiments, the phosphine modifiedbiomolecule is a phosphine modified glycoprotein.

In certain embodiments, the azido modified biomolecule is attached to asolid support using functional groups other than azide reactivefunctional groups, whereupon the attached azido modified biomolecule isused to form a conjugate under click chemistry conditions wherein thereporter molecule, carrier molecule or another solid support comprises aterminal alkyne. In another embodiment the azido modified biomolecule isattached to a solid support using functional groups other than azidereactive functional groups, whereupon the attached azido modifiedbiomolecule is used to form a conjugate under Staudinger ligationconditions wherein the reporter molecule, carrier molecule or othersolid support comprises a triaryle phosphine. In certain embodiments, anazido modified glycoprotein is attached to a solid support usingfunctional groups other than azide reactive functional groups, whereuponthe attached azido modified glycoprotein is used to form a conjugateunder click chemistry conditions wherein the reporter molecule, carriermolecule or another solid support comprises a terminal alkyne. Inanother embodiment the azido modified glycoprotein is attached to asolid support using functional groups other than azide reactivefunctional groups, whereupon the attached azido modified glycoprotein isused to form a conjugate under Staudinger ligation conditions whereinthe reporter molecule, carrier molecule or another solid supportcomprises a triaryl phosphine.

In another aspect is provided a method for detecting immobilized azidomodified biomolecules, wherein the method includes the following:

-   -   a) immobilizing the azido modified biomolecules on a solid or        semi-solid atrix to form an immobilized azido modified        biomolecule;    -   b) contacting the immobilized azido modified biomolecule with a        reporter molecule that contains an alkyne reactive group to form        a contacted azido modified biomolecule;    -   c) incubating the contacted azido modified biomolecule for a        sufficient amount of time to form a reporter        molecule-biomolecule conjugate;    -   d) illuminating the reporter molecule-biomolecule conjugate with        an appropriate wavelength to form an illuminated reporter        molecule-biomolecule conjugate, and    -   e) observing the illuminated reporter molecule-biomolecule        conjugate whereby the immobilized azido modified biomolecule is        detected.        In certain embodiments the azido modified biomolecule is an        azido modified protein, while in other embodiments the azido        modified biomolecule is an azido modified glycoprotein.

In another aspect is provided a method for detecting immobilized alkynemodified biomolecules, wherein the method includes the following:

-   -   a) immobilizing the alkyne modified biomolecules on a solid or        semi-solid atrix to form an immobilized alkyne modified        biomolecule;    -   b) contacting the immobilized alkyne modified biomolecule with a        reporter molecule that contains an azide reactive group to form        a contacted alkyne modified biomolecule;    -   c) incubating the contacted alkyne modified biomolecule for a        sufficient amount of time to form a reporter        molecule-biomolecule conjugate;    -   d) illuminating the reporter molecule-biomolecule conjugate with        an appropriate wavelength to form an illuminated reporter        molecule-biomolecule conjugate, and    -   e) observing the illuminated reporter molecule-biomolecule        conjugate whereby the immobilized alkyne modified biomolecule is        detected.        In certain embodiments the alkyne modified biomolecule is an        alkyne modified protein, while in other embodiments the alkyne        modified biomolecule is an alkyne modified glycoprotein.

Samples and Sample Preparation

The end user will determine the choice of the sample and the way inwhich the sample is prepared. Samples that can be used with the methodsand compositions described herein include, but are not limited to, anybiological derived material or aqueous solution that contains abiomolecule. In certain embodiments, a samples also includes material inwhich a biomolecule has been added. The sample that can be used with themethods and compositions described herein can be a biological fluidincluding, but not limited to, whole blood, plasma, serum, nasalsecretions, sputum, saliva, urine, sweat, transdermal exudates,cerebrospinal fluid, or the like. In other embodiments, the sample arebiological fluids that include tissue and cell culture medium whereinbiomolecule of interest has been secreted into the medium. Cells used insuch cultures include, but are not limited to, prokaryotic cells andeukaryotic cells that include primary cultures and immortalized celllines. Such eukaryotic cells include, without limitation, ovary cells,epithelial cells, circulating immune cells, β cells, hepatocytes, andneurons. In certain embodiments, the sample may be whole organs, tissueor cells from an animal, including but not limited to, muscle, eye,skin, gonads, lymph nodes, heart, brain, lung, liver, kidney, spleen,thymus, pancreas, solid tumors, macrophages, mammary glands,mesothelium, and the like.

Various buffers can be used in the methods described herein, includinginorganic and organic buffers. In certain embodiments the organic bufferis a zwitterionic buffer. By way of example only, buffers that can beused in the methods described herein include phosphate buffered saline(PBS), phosphate, succinate, citrate, borate, maleate, cacodylate,N-(2-Acetamido)iminodiacetic acid (ADA), 2-(N-morpholino)-ethanesulfonicacid (MES), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES),piperazine-N,N′-2-ethanesulfonic acid (PIPES),2-(N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO),N,N-bis-(hydroxyethyl)-2-aminoethanesulfonic acid (BES),3-(N-morpholino)-propanesulfonic acid (MOPS),N-tris-(hydroxymethyl)-2-ethanesulfonic acid (TES),N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid (HEPES),3-(N-tris-(hydroxymethyl)methylamino)-2-hydroxypropanesulfonic acid(TAPSO), 3-(N,N-Bis[2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid(DIPSO), N-(2-Hydroxyethyl)piperazine-N′-(2-hydroxypropanesulfonic acid)(HEPPSO), 4-(2-Hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS),N-[Tris(hydroxymethyl)methyl]glycine (Tricine),N,N-Bis(2-hydroxyethyl)glycine (Bicine),(2-Hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]-1-propanesulfonic acid(TAPS), N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonicacid (AMPSO), tris(hydroxy methyl)amino-methane (Tris),TRIS-Acetate-EDTA (TAE), glycine,bis[2-hydroxyethyl]iminotris[hydroxymethyl]methane (BisTris), orcombinations thereof. In certain embodiments, wherein such buffers areused in gel electrophoresis separations the buffer can also includeethylene diamine tetraacetic acid (EDTA).

The concentration of such buffers used in the methods described hereinis from about 0.1 mM to 1 M. In certain embodiments the concentration isbetween 10 mM to about 1 M. In certain embodiments the concentration isbetween about 20 mM and about 500 mM, and in other embodiments theconcentration is between about 50 mM and about 300 mM. In certainembodiments, the buffer concentration is from about 0.1 mM to about 50mM, while in other embodiments the buffer concentration if from about0.5 mM to about 20 mM.

The pH will vary depending upon the particular assay system, generallywithin a readily determinable range wherein one or more of the sulfonicacid moieties is deprotonated.

In certain embodiments, buffers used in the methods described hereinhave a pH between 5 and 9 at ambient temperature. In certain embodimentsthe buffer has a pH between 6 and 8.5 at ambient temperature. In certainembodiments the buffer has a pH between 6 and 8 at ambient temperature.In certain embodiments the buffer has a pH between 6 and 7 at ambienttemperature. In certain embodiments the buffer has a pH between 5 and 9at 25° C. In certain embodiments the buffer has a pH between 6 and 8.5at 25° C. In certain embodiments the buffer has a pH between 6 and 8 at25° C. In certain embodiments the buffer has a pH between 6 and 7 at 25°C.

In certain embodiments, the sample used in the methods described hereinhave a non-ionic detergent to the sample. Non-limiting examples of suchnon-ionic detergents added to the samples used in the methods describedherein are polyoxyalkylene diols, ethers of fatty alcohols includingalcohol ethoxylates (Neodol from Shell Chemical Company and Tergitolfrom Union Carbide Corporation), alkyl phenol ethoxylates (Igepalsurfactants from General Aniline and Film Corporation), ethyleneoxide/propylene oxide block copolymers (PLURONIC™ Series from BASFWyandotte Corporation), polyoxyethylene ester of a fatty acids (StearoxCD from Monsanto Company), alkyl phenol surfactants (Triton series,including Triton X-100 from Rohm and Haas Company), polyoxyethylenemercaptan analogs of alcohol ethoxylates (Nonic 218 and Stearox SK fromMonsanto Company), polyoxyethylene adducts of alkyl amines (Ethoduomeenand Ethomeen surfactants from Armak Company), polyoxyethylene alkylamides, sorbitan esters (such as sorbitan monolaurate) and alcoholphenol ethoxylate (Surfonic from Jefferson Chemical Company, Inc.).Non-limiting examples of sorbitan esters include polyoxyethylene(20)sorbitan monolaurate (TWEEN20), polyoxyethylene(20) sorbitanmonopalmitate (TWEEN40), polyoxyethylene(20) sorbitan monostearate(TWEEN60) and polyoxyethylene(20) sorbitan monooleate (TWEEN 80). Incertain embodiments, the concentration of such non-ionic detergentsadded to a sample is from 0.01 to 0.5%. In other embodiments theconcentration is from about 0.01 to 0.4 vol. %. In other embodiments theconcentration is from about 0.01 to 0.3 vol. %. In other embodiments theconcentration is from about 0.01 to 0.2 vol. %. In other embodiments theconcentration is from about 0.01 to 0.1 vol. %.

Illumination

The compounds and compositions described herein may, at any time before,after or during an assay, be illuminated with a wavelength of light thatresults in a detectable optical response, and observed with a means fordetecting the optical response. In certain embodiments, suchillumination can be by a violet or visible wavelength emission lamp, anarc lamp, a laser, or even sunlight or ordinary room light, wherein thewavelength of such sources overlap the absorption spectrum of afluorophore or chromaphore of the compounds or compositions describedherein. In certain embodiments, such illumination can be by a violet orvisible wavelength emission lamp, an arc lamp, a laser, or even sunlightor ordinary room light, wherein the fluorescent compounds, includingthose bound to the complementary specific binding pair member, displayintense visible absorption as well as fluorescence emission.

In certain embodiments, the sources used for illuminating thefluorophore or chromaphore of the compounds or compositions describedherein include, but are not limited to, hand-held ultraviolet lamps,mercury arc lamps, xenon lamps, argon lasers, laser diodes, blue laserdiodes, and YAG lasers. These illumination sources are optionallyintegrated into laser scanners, flow cytometer, fluorescence microplatereaders, standard or mini fluorometers, or chromatographic detectors.These fluorescence emission of such fluorophores is optionally detectedby visual inspection, or by use of any of the following devices: CCDcameras, video cameras, photographic film, laser scanning devices,fluorometers, photodiodes, photodiode arrays, quantum counters,epifluorescence microscopes, scanning microscopes, flow cytometers,fluorescence microplate readers, or by means for amplifying the signalsuch as photomultiplier tubes. Where the sample is examined using a flowcytometer, a fluorescence microscope or a fluorometer, the instrument isoptionally used to distinguish and discriminate between the fluorescentcompounds of the invention and a second fluorophore with detectablydifferent optical properties, typically by distinguishing thefluorescence response of the fluorescent compounds of the invention fromthat of the second fluorophore. Where a sample is examined using a flowcytometer, examination of the sample optionally includes isolation ofparticles within the sample based on the fluorescence response by usinga sorting device.

In certain embodiments, fluorescence is optionally quenched using eitherphysical or chemical quenching agents.

Kits of the Invention

In another aspect, the present invention provides kits that includeUDP-GalNAz, a GalT enzyme; an azide reactive reporter molecule, carriermolecule or solid support.

In one aspect, the invention includes a kit for labeling a biomoleculethat includes at least one label that comprises a terminal alkyne, asolution comprising copper, and a solution that comprises a copper (I)chelator. The kit can further comprise a solution that comprises areducing agent, one or more buffers, or one or more detergents.

In one embodiment, an alkyne label provided in a kit is a fluorophore,such as, but not limited to, a xanthene, coumarin, borapolyazaindacene,pyrene and cyanine. In one embodiment, a kit provides two or moredifferent terminal alkyne-containing labels one or more of which is afluorophore, In other embodiments, an alkyne label provided in a kit isa tag, such as but not limited to a peptide or a hapten, such as biotin.

In preferred embodiments, a copper (I) chelator provided in the kit is a1,10 phenanthroline, preferably bathocuproine disulfonic acid. In someembodiments, copper is provided in the form of a copper sulfate orcopper acetate solution. In some embodiments, a reducing agent isprovided in the form of ascorbate.

In another aspect, the invention includes a kit for labeling abiomolecule that includes at least one label that comprises an azidogroup, a solution comprising copper, an a solution that comprises acopper (I) chelator. The kit can further comprise a solution thatcomprises a reducing agent, one or more buffers, or one or moredetergents.

In one embodiment of this aspect, an azido-containing label provided ina kit is a fluorophore, such as, but not limited to, a xanthene,coumarin, borapolyazaindacene, pyrene and cyanine. In other embodiments,an azido label provided in a kit is a tag, such as but not limited to apeptide or a hapten, such as biotin.

In one embodiment, a kit provides two or more different azido-containinglabels one or more of which is a fluorophore, In preferred embodiments,a copper (I) chelator provided in the kit is a 1,10 phenanthroline,preferably bathocuproine disulfonic acid. In some embodiments, copper isprovided in the form of a copper sulfate or copper acetate solution. Insome embodiments, a reducing agent is provided in the form of ascorbate.

In other embodiments, a kit can further include one or more reagents andsolutions for chromogenic detection on Western blots.

A detailed description of the invention having been provided above, thefollowing examples are given for the purpose of illustrating theinvention and shall not be construed as being a limitation on the scopeof the invention or claims.

EXAMPLES Example 1 Synthesis of UDPGalNAz

The synthesis of UDPGalNAz is shown in the following reaction scheme.

Azidoacetic Acid.

To a solution of iodoacetic acid (8.0 g, 43.0 mmol) and H₂O (100 mL) wasadded sodium azide (5.62 g, 86.0 mmol). The solution was stirred at RTand protected from light. After 4 days, the solution was diluted with 1N HCl (30 mL) and the pH was check to ensure that it was in the range of2-3. The solution was extracted with EtOAc (2×100 mL), then the combinedorganics were washed with saturated NaHSO₃ (1×50 mL), brine (1×50 mL)and dried over MgSO₄. The solution was decanted, concentrated and thecrude azidoacetic acid (3.34 g, 53%) was used directly in the next stepwithout further purification.

N-Azidoacetylgalactosamine (mix of anomers)

To a solution of azidoacetic acid (3.34 g, 33.06 mmol) in methanol (170mL) was added D-galactosamine hydrochloride (5.09 g, 23.62 mmol)followed by triethylamine (7.90 mL, 56.68 mmol). This solution wasstirred at RT for 5 min and then cooled to 0° C. 1-Hydroxybenzotriazole(3.19 g, 23.62 mmol) was added followed byN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (9.05 g,47.24 mmol). The ice was allowed to melt on its own and the clear, paleorange reaction solution was stirred overnight at RT, and protected fromlight. The honey-colored solution was concentrated to yield a yellowresin. The crude yellow material (mixture of 2 anomers, assumed 100%conversion) was used directly in the next reaction without furtherpurification.

1,3,4,6-Tetra-O-acetyl-N-azidoacetylgalactosamine (Ac₄GalNAz, mix ofanomers)

To a solution of N-azidoacetylgalactosamine (mix of anomers, 23.62 mmol)in pyridine (140 mL) was added acetic anhydride (70 mL). The cloudy,orange/brown colored solution was stirred at RT overnight and protectedfrom light. The solution was concentrated to yield a brown, viscous oil.The crude oil was purified via silica gel loaded with 33-50%EtOAc/hexanes, affording 4.35 g (60%, mixture of 2 anomers) of theproduct.

3,4,6-Tri-O-acetyl-N-azidoacetylgalactosamine (mix of anomers)

To a solution of 1,3,4,6-tetra-O-acetyl-N-azidoacetylgalactosamine (mixof anomers, 1.60 g, 3.75 mmol) in THF (20 mL) was added benzylamine(0.49 mL, 4.5 mmol). The solution was heated in an oil bath at 50° C.and protected from light. After 15 hr, the initial clear, canary yellowsolution turned brown in color. The solution was removed from the oilbath and concentrated. The crude was purified via silica columnchromatography (50-100% EtOAc/hexanes) to afford the product (1.18 g,82%, brown oil, mixture of 2 anomers).

Diallyl(3,4,6-tetra-O-acetyl-2-azidoacetamido-2-dexocy-D-galactopyranosylphosphate

To a solution of 3,4,6-tri-O-acetyl-N-azidoacetylgalactosamine (mix ofanomers, 0.34 g, 0.88 mmol) in CH₂Cl₂ (9.0 mL) was added 1H-tetrazole(0.31 g, 4.41 mmol). The reaction was stirred at RT for 10 min andprotected from light. Diallyldiisopropylphosphoramidite (0.70 mL, 2.64mmol) was added dropwise and the reaction was stirred at RT for 3 h. Thesolution was subsequently cooled to −40° C. in an acetonitrile/dry icebath and 3-chloroperbenzoic acid (0.76 g, 4.41 mmol) and stirred at −40°C. for 10 min. The solution was then allowed to warm to RT slowly over 1h (transferred from a −40° C. bath to 0° C. bath packed with ice). After1 hr, ice was still present so removed ice bath and let stir at RT for20 min. The reaction solution was diluted with CH₂Cl₂ (45 mL), washedsequentially with 10% aqueous Na₂SO₃ (2×45 mL), saturated NaHCO₃ (2×45mL), and H₂O (2×45 mL). The organic layer was then dried over Na₂SO₄,decanted and concentrated. The crude material was purified via silicagel column chromatography (60-100% EtOAc/hexanes) to afford the product,(0.18 g, 39%).

UDPGalNAz.

To a solution of diallyl(3,4,6-tetra-O-acetyl-2-azidoacetamido-2-dexocy-D-galactopyranosylphosphate (0.10 g, 0.18 mmol) and THF:MeOH (1:1, 2.1 mL:2.1 mL) underargon was added p-toluenesulfonic acid, sodium salt (0.14 g, 0.72 mmol)followed by tetrakis(triphenylphosphine)palladium(0) (30 mg, 0.026mmol). The solution was protected from light and stirred at RT for 3 h.After 4 days, the reaction solution was concentrated in vacuo andco-evaporated with toluene (3×20 mL). The crude material was suspendedin CH₃CN (0.86 mL) and triethylamine (0.27 mL) was added. The solutionwas cooled to 0° C. and freshly prepared UMP-N-methylimidazole (0.22mmol) in CH₃CN (1.7 mL) was added dropwise. The resulting yellowsolution was stirred at 0° C. for 3 h. The reaction solution wasconcentrated, then resuspended in a solution of MeOH:H₂O:triethylamine(5:2:1, 10 mL) and stirred at RT for 20 h. The solution was concentratedin vacuo to yield a crude yellow syrup. This material was dissolved inH₂O (15 mL) and extracted with CH₂Cl₂ (3×50 mL) to remove organicbyproducts. The organic layer was discarded and the aqueous layer wasconcentrated and co-evaporated with toluene. The resulting viscous, paleorange, clear oil was obtained. The crude material was dissolved in 100mM NH₄HCO₃ degassed buffer (2.0 mL). Because the solution was cloudy inappearance and not clear, it was filtered through a Acrodisc PF 0.8/0.2mM Supor, sterile, single use, non-pyrogenic filter from GelmanSciences, prod #4187. The clear, pale orange solution obtained wasloaded onto a BioGel P2, extra fine column (1.5 cm×80 cm) and flashed ata flow rate of 0.13 mL/min. The column was run overnight with 100 mMNH₄HCO₃ buffer; 2.6 mL per fraction were collected. The fractionscontaining product were collected (TLC 5:3:1, EtOH, NH₄OH, H₂O,R_(f)=0.70, UV active). After combining the product-containingfractions, the solution was concentrated and the resulting white solidwas dissolved in a minimal amount of water and passed through a Dowex(BioRad AG 50W-X8-200 resin, purchased in the sodium form). All of theeluent was collected and concentrated after the UV active materialceased eluting from the column. A pale, tan foam was obtained. Thismaterial was dissolved in minimal water and lyophilized to afford theproduct (0.10 g, 83%) as a tan, crystalline material.

Example 2 Synthesis of UMP-N-methylimidazolide

The synthesis of UMP-N-methylimidazolide is shown in the followingreaction scheme.

Uridine 5′-monophosphate triethylammonium salt (5′-UMP triethylammoniumsalt)

Uridine 5′-monophosphate disodium salt (1.0 g) was dissolved in H₂O (2ml) and passed through Dowex resin triethylammonium salt (2.7 cm×7.0 cmcolumn size) with H₂O as eluent. All of the eluent was concentrated andafter the UV active material ceased eluting from the column. Thesolution was concentrated and co-evaporated with toluene to until awhite, crystalline solid was obtained (0.78 g, 80%).

Uridine monophosphate-N-methylimidazole (UMP-N-methylimidazole)

To a suspension of uridine 5′-monophosphate triethylammonium salt (0.097g, 0.22 mmol) in CH₃CN (0.70 mL) was added N,N-dimethylaniline (0.11 mL,0.864 mmol), and triethylamine (0.03 mL, 0.22 mmol). The suspension wascooled to 0° C. In a separate flask, trifluoroacetic anhydride (0.15 mL,1.08 mmol) was added to CH₃CN (0.21 mL) and cooled to 0° C. Thissolution was added dropwise via microsyringe, to the suspension ofuridine 5′-monophosphate triethylammonium salt at 0° C.; a yellow, clearsolution was obtained. After 5 min, the solution was removed from theice bath and stirred at RT for 30 min. The solution was concentrated invacuo and placed under dry argon. The mixed phosphoryl anhydridesolution was then cooled to 0° C. In a separate flask methylimidazole(0.051 mL, 0.65 mmol) was added to a solution of CH₃CN (0.2 mL) andtriethylamine (0.15 mL, 1.08 mmol). The resulting clear and colorlesssolution was cooled to 0° C. and subsequently added dropwise to themixed phosphoryl anhydride solution at 0° C. The solution was thenstirred at 0° C. for 5-10 min. The yellow color darkened and after 20min and the TLC (10:10:1, CHCl₃:MeOH:1 mM NH₄OAc pH 7), showed completeconversion to product (R_(f)=0.28, UV active). CH₃CN (1.7 mL) was addedto the solution at 0° C., and this crude material was used directly inthe next step; 100% conversion is assumed.

Example 3 Synthesis of Dapoxyl® Alkyne

The synthesis of Dapoxyl® alkyne is shown in the following reactionscheme.

To a solution of Dapoxyl® carboxylic acid, succinimidyl ester (50 mg,0.12 mmol) in DMF (0.4 mL) at RT was added propargylamine (42 μL, 0.61mmol). The initial clear orange solution turned yellow and cloudy. After˜15 min at RT the reaction was complete, and the solution wasconcentrated to dryness. The residue was purified via HPLC to afford theproduct (36 mg, 84%). TLC (10% EtOAc, CHCl₃) R_(f)=0.30; ESI m/z 346(M⁺, C₂₁H₁₉N₃O₂ requires 346).

Example 4 Synthesis of 5-Carboxytetramethyl rhodamine alkyne(5-TAMRA-alkyne)

The synthesis of 5-Carboxytetramethyl rhodamine alkyne (5-TAMRA-alkyne)is shown in the following reaction scheme.

To a solution of 5-carboxytetramethyl rhodamine, succinimidyl ester(5-TAMRA-SE, 0.10 g, 0.19 mmol) in DMF (0.5 mL) was added propargylamine(25 μL, 0.38 mmol) and H₂O (0.5 mL). After stirring the solution for 30min at RT, the solution was concentrated in vacuo. Purification via HPLC(Phenomenex Prodigy ODS, internal diameter 21.2 mm, eluent 25-40% CH₃CNin 25 mM TEAA, pH 4.7, flow rate of 15 mL/min) gave 68 mg of product(82%, a purple solid) t_(R)=23-33 min. TLC (CH₃CN: H₂O, 8:2) R_(f)=0.67.

Example 5 Synthesis of Biotin Alkyne

The synthesis of Biotin alkyne is shown in the following reactionscheme.

To a solution of EZ-link NHS-PEO₄-biotin (25 mg, 0.004 mmol, Pierce) inDMF (0.1 mL) was added propargylamine (0.1 mL). After stirring thesolution for 90 min at RT, some starting material was still seen.Additional propargylamine (0.2 mL) was added and the solution wasstirred for another 60 min. The solution was concentrated in vacuo. Thecrude material was purified via HPLC to afford 14.4 mg (64%) of theproduct as a yellow solid. TLC (CHCl₃: MeOH, 7:1) R_(f)=0.23; ESI m/z529 (M⁺, C₂₄H₄₀N₄O₇S requires 529).

Example 6 Synthesis of Compound 1

The synthesis of Compound 1 is shown in the following reaction scheme.

To a solution of Alexa Fluor® 488 carboxylic acid, succinimidyl ester,dilithium salt (mixed isomers, 50 mg, 0.079 mmol) in DMF (2.0 mL) wasadded propargylamine (54 L, 0.79 mmol). The solution was stirredovernight at RT. The initial deep red solution turned pale yellow incolor and became clear. The solution was concentrated in vacuo andpurified via silica gel thin layer chromatography (prep plate, 20% H₂O,CH₃CN) to afford the product (20 mg, 44%) as an orange solid. TLC (3:1,CH₃CN:H₂O) R_(f)=0.70; ESI neg m/z 570 (M⁺, C₂₄H₁₆N₃O₁₀S²⁻ requires570).

Example 7 Synthesis of Compound 2

The synthesis of Compound 2 is shown in the following reaction scheme.

To a solution of Alexa Fluor® 532 carboxylic acid, succinimidyl ester(50 mg, 0.070 mmol) in DMF (2.2 mL) was added propargylamine (100 L,1.46 mmol). The solution was stirred overnight at RT. H₂O (1.0 mL) wasadded to the solution and the solution was stirred an additional hour.The solution was concentrated in vacuo and the crude material waspurified via HPLC to afford the product (30 mg, 65%). TLC (8:2,CH₃CN:H₂O) R_(f)=0.58; ESI m/z 664 (M⁺, C₃₃H₃₃N₃O₈S₂ requires 664).

Example 8 Synthesis of Compound 3

The synthesis of Compound 3 is shown in the following reaction scheme.

To a solution of Alexa Fluor® 633 carboxylic acid, succinimidyl ester,bis(triethylammonium salt) (50 mg, 0.041 mmol) in DMF (2.0 mL) was addedpropargylamine (28 μL, 0.40 mmol). The solution was stirred overnight atRT. H₂O (1.0 mL) was added to the solution and the solution was stirredan additional hour. The solution was concentrated in vacuo and theproduct (39 mg, 99%). TLC (8:2, CH₃CN:H₂O) R_(f)=0.66; ESI m/z 963 (M⁺,C₄₀H₃₄F₂N₃O₁₁S₆ requires 963).

Example 9 Synthesis of Triarylphosphine-TAMRA Dye for StaudingerLigation

The synthesis of triarylphosphine-TAMRA dye is shown in the reactionscheme below.

To a solution of acid 1 (Science 2000, 287, 2007-2010) (80 mg, 0.26mmol) in CH₂Cl₂ (5 mL) was addedN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDCI, 75mg, 0.39 mmol) and N-hydroxysuccinimide (NHS, 5 mg). The solution wasstirred at RT. After 2.5 h, amine 2 (50 μL, 0.26 mmol) was added and thesolution was stirred overnight. The solution was partitioned betweenCHCl₃ (15 mL) and H₂O (5 mL). The organic layer was separated and theaqueous layer was reextracted with CHCl₃ (15 mL). The combined organiclayers were rinsed once with H₂O (5 mL), followed by saturated aqueousNaCl (5 mL). The organic layer was dried over Na₂SO₄, decanted andconcentrated. The crude was purified via chromatography (silica, 2%MeOH, CHCl₃) to afford the product (99 mg, 71%) as a clear, yellow oil.

To a solution of 3 (10 mg, 0.018 mmol) in CH₂Cl₂ (1.0 mL) was addedtrifluoracetic acid (TFA, 0.5 mL) and the solution was stirred at RT.After 30 min, the solution was concentrated and reevaporated fromtoluene (2×2 mL). The residue (4, 0.018 mmol) was dissolved in DMF (0.2mL) and N-ethyldiisopropylamine (DIEA, 12 uL, 0.72 mmol), and5-carboxytetramethyl rhodamine, succinimidyl ester (5-TAMRA-SE, 9 mg,0.022 mmol) were added. The solution was stirred at RT for 2.5 h,concentrated and purified via silica gel (prep plate, 20% H₂O in CH₃CN)to afford the product (7.4 mg, 48%). TLC (20% H₂O in CH₃CN)R_(f)=0.23;ESI m/z 529 (M⁺, C₂₄H₄₀N₄O₇S requires 529).

Example 10 Synthesis of Triarylphosphine-Biotin for Staudinger Ligation

The synthesis of triarylphosphine-biotin is shown in the followingreaction scheme.

To a solution of 3 (5.3 mg, 0.010 mmol) in CH₂Cl₂ (1.2 mL) was addedtrifluoracetic acid (TFA, 0.5 mL) and the solution was stirred at RT.After 2 h, the solution was concentrated and reevaporated from toluene(2×2 mL). The residue (4, 0.010 mmol) was dissolved in DMF (0.1 mL) andN-ethyldiisopropylamine (DIEA, 3 μL, 0.02 mmol), and EZ-linkNHS-PEO₄-biotin (7 mg, 0.012 mmol) were added. The solution was stirredat RT for 1 h, quenched with saturated NH₄ ⁺Cl⁻ and partitioned betweenCHCl₃ (10 mL) and H₂O (1 mL). The aqueous layer was extracted repeatedlywith CHCl₃ (10 mL per extraction) until no ultraviolet spot was observedby TLC. The combined organic layers were concentrated and purified viasilica gel (prep plate, 7:1 CHCl₃:MeOH) to afford the product (2.2 mg,25%). TLC (7:1 CHCl₃:MeOH, developed 3 times) R_(f)=0.50; ESI m/z 909(M+H⁺, C₄₆H₆₃N₅O₁₀PS requires 909).

Example 11 Metabolic Labeling and “Click” Detection of GlycoproteinSubclasses

Jurkat cells were fed 40 μM Ac₄ManNAz or Ac₄GalNAz for 3 days (A) or 250μM Ac₄GlcNAz overnight (B). Harvested cells were sonicated in 50 mM Trisbuffer, pH 8.0 with protease and phosphatase inhibitors, and the lysateswere subjected to high-speed centrifugation (100K×g). The membranepellet proteins from ManNAz- and GalNAz-treated cells, and the solublesupernatant cells from the GlcNAc-treated cells, were precipitated withchloroform/methanol, dissolved in detergent, and labeled with afluorescent alkyne probe in the presence of 1 mM CuSO4, and 5 mMascorbic acid (1). 10 μg of the labeled, precipitated proteins were runon 1-D NuPAGE® Novex® 4-12% gels (Invitrogen). Images were obtained onthe Fuji FLA-3000 scanner (Fuji) using 532 nm excitation. Gels were thenpost stained with SYPRO® Ruby protein stain (Invitrogen, Carlsbad,Calif.) and imaged using excitation at 473 nm. Control lanes representextracts from unfed cells but treated with the fluorescent probe. SeeFIG. 2.

Example 12 Separation of Ac₄GlcNAz-Treated Soluble Jurkat Cell Proteinsby 2-D Gels

Jurkat cells were cultured overnight with 250 μM Ac₄GlcNAz. Solublelysate proteins were prepared as above using sonication andultracentrifugation and labeled for 1 hour with a fluorescent alkyneprobe. 40 μg of the labeled proteins were precipitated and resolubilizedin 7M urea, 2M thiourea, 65 mM DTT, 2% CHAPS, 1% Zwittergent 3-10, 1% pH3-10 carrier ampholytes and separated on pH 3-10 IEF strips in the firstdimension and 4-12% Bis-Tris gels with MOPS buffer in the seconddimension. See FIG. 3.

Example 13 In Gel Detection of 40 and 50 kD Azide-Labeled Model Proteins

25 pmols each of 40 and 50 kD model proteins with single N-terminalazides were spiked into 100 μg of Jurkat cell lysates (upper panels), ornot (lower panels). Proteins were labeled with a fluorescent alkyneprobe, serially diluted as shown, and run on NuPAGE® Novex® 4-12% gels.Images (left panels) were obtained on the FLA-3000 scanner using 532 nmexcitation. Gels were then post stained with SYPRO® Ruby stain andimaged using excitation at 473 nm (right panels). Detection sensitivityof the labeled proteins is less than 10 femtomoles. See FIG. 4.

Example 14 Labeling Efficiency of 40 and 50 kd Azide-Labeled ModelProteins is Unchanged in Complex Protein Extracts

Either 100 ng (25 pmol) or 10 ng (2.5 pmol) each of azide-labeled 40 Kd& 50 Kd proteins were labeled with fluorescent alkyne probe as above ina background of either 100, 50, 25, or 0 μg of control Jurkat lysate(left panel). Note: 100 μg of control lysate was added after labeling tothe ‘0 lysate’ to facilitate recovery of the labeled protein byprecipitation. The gel was post stained with SYPRO® Ruby total proteinstain. See FIG. 5.

Example 15 Selective Analysis of Cell Surface Versus Total GlycoproteinSubclasses by Gel Electrophoresis

HeLa cells were fed Ac₄GalNAz or Ac₄ManNAz sugars for 48 hours. For cellsurface glycoprotein analysis, the surfaces of live cells were labeledwith dye-alkyne, lysed, and the purified proteins were analyzed by gelelectrophoresis (lanes 2 and 5, see legend). For total glycoproteinsubclass analysis, cells were lysed, the purified proteins were labeledin solution, and ran on the gel (lanes 3 and 6). Control unfed cells areshown in gel lanes 1 and 4.20 g of protein was loaded per gel lane. TheGalNAz incorporated proteins in lane 2 represent cell surface O-linkedglycoproteins only, whereas total GalNAz metabolically labeledglycoproteins are shown in lane 3. These proteins represent both cellsurface O-linked glycoproteins, intracellular GalNAz labeled proteinslocated in golgi apparatus and transport vesicles, and O-GlcNAc modifiedproteins (GalNAz was recently shown to flux into the O-GlcNAcbiosynthetic pathway). Lane 5 represents ManNAz labeling of cell surfacesialic acid-containing glycoproteins, whereas lane 6 represents labelingof both cell surface sialic acid-containing glycoproteins andintracellular sialic acid-containing glycoproteins located in golgiapparatus and transport vesicles.

Example 16 Metabolic Labeling of Glycoprotein Subclasses in Live Animalsand in Gel Fluorescence Detection of Labeled Glycoproteins

Metabolic labeling of O-linked and sialic acid-containing glycoproteinswas accomplished by administration of the unnatural tetraacetylatedazide-modified sugar precursors, Ac₄GalNAz or Ac₄ManNAz, respectively(300 mg/kg in 70% DMSO), to activated Her2/Neu transgenic mice over a 7day regimen. After 7 days the mice were sacrificed and various organtissues were dissected, lysed, and protein extracts were reacted with 10mM fluorescent TAMRA alkyne in Tris Buffer, pH 8.0, with 2 mM CuSO4, 20mM ascorbate, 5 mM BCS, and 25% propylene glycol. Labeled glycoproteinswere precipitated, dissolved in 1-D SDS-PAGE buffer, analyzed by gelelectrophoresis, and detected by fluorescence laser scanning. Afterscanning the gels were post-stained with SYPRO® Ruby total protein stainto view total proteins. The upper panels below show 1-D gels of labeledtissue proteins from heart muscle (H), liver (L), or kidney (K). Lowerpanels show same gels after post-staining with SYPRO® Ruby total proteinstain.

Example 17 Fluorescent “Click” Labeling of Live and Fixed Cells:Selective Labeling of Cell Surface Versus Internal Glycoproteins

Jurkat cells were fed 30 μM Ac₄HexNAz sugars, ManNAz, GalNAz, or GlcNAz,for 3 days. In A., live Jurkat cells were labeled with 10 mM Alexa Fluor488 alkyne probe for 30 minutes in the presence of Tris buffer, pH 8.0,2 mM CuSO4, 20 mM ascorbate, and 5 mM BCS. The cells were then fixedwith 4% formaldehyde/PBS for 15 min and analyzed by flow cytometry. InB, cells were fixed and permeabilized, before labeling under the sameconditions as in A, and analyzed by flow cytometry. Theses resultsdemonstrate the ability to selectively stain cell surface glycoproteinsonly, or cell surface and intracellular glycoproteins. SignificantGlcNAz staining is apparent only after cell permeabilization, as themodification is specific to the inside of the cell.

Example 18 Enzymatic Labeling and Detection of α-Crystallin O-GlcNAc

α-crystallin O-GlcNAc was enzymatically labeled with azide (UDP-GalNAz)using a modified b-GalT1 enzyme. The protein was subsequently reactedwith a fluorescent alkyne probe as described. The proteins were run on1-D NuPAGE® Novex® 4-12% gels at the dilutions shown. Note: Only 2-10%of α-crystallin is O-GlcNAc-modified and therefore the detectionsensitivity of the O-GlcNAc moiety is in the mid-to-low femtomole range(10-45 fmols). See FIG. 6.

Example 19 Comparison of GalT1 Enzyme Labeling with a-O-GlcNAcMonoclonal Antibody CTD 110.6

In A., α-crystallin was enzymatically labeled with the modified GalT1enzyme and subsequently reacted with a biotin-alkyne probe. The proteinswere run on 1-D NuPAGE® Novex® 4-12% MES gels, at the dilutions shown,and blotted onto PVDF membrane. The PVDF membrane was then incubated instreptavidin-HRP and proteins were detected using ECL Plus™ (GEBiosystems). Lane 2 (NE) represents the 8 pmoL no-enzyme added control.Note: In A, the detection sensitivity of O-GlcNAc by Western blot is inthe low femtomole range (3-10 fmols). In B., untreated α-crystallin wasrun on 1-D gels and blotted as described above. The PVDF membrane wasprocessed using the O-GlcNAc Western Blot Detection Kit (Pierce)according to manufacturer's instructions. The kit utilizes the CTD110.6α-O-GlcNAc monoclonal antibody. Lane 2 contains 5 ng of the positivecontrol (O-GlcNAc-modified BSA) provided in the kit. No α-crystallin isdetected using the antibody detection system. See FIG. 7.

Example 20 Multiplex Detection of O-GlcNAc Proteins, Phosphoproteins andTotal Proteins in the Same 2-D Gel

Soluble extracts from GlcNAz-fed Jurkat cells were labeled with UVexcitable alkyne dye for 2 hours. The chloroform/methanol precipitatedproteins were run on 2-D gels as described previously. The gel wasrinsed in water and imaged with UV transillumination and 600/bp emissionon a Lumi-Imager™ (Roche). The gel was then stained with Pro-Q® Diamondphosphoprotein stain, imaged with 532 nm excitation/580 LP emission on aFLA-3000 laser imager, stained with SYPRO® Ruby total protein stain, andimaged again with 473 nm excitation and 580 nm longpass emissionaccording to the manufacturer's instructions. See FIG. 8.

Example 21 Multiplexed Western Blot Detection of O-GlcNAc and Cofilin

25 μg of soluble Jurkat cell proteins from Example 12 were run on 2-Dgels as described and blotted onto PVDF membrane. The PVDF membrane wasincubated in α-cofilin polyclonal Ab and detected with GAR-HRP secondaryAb with ECL Plus™ detection (GE Biosystems). After imaging, the blot wasincubated in streptavidin AP and O-GlcNAc proteins were detected usingthe WesternBreeze® chemiluminescent detection kit (Invitrogen). See FIG.9.

Example 22 Differential Detection of O-GlcNAc Modified Proteins inControl and Inhibitor-Treated Cultured Cell Extracts

Jurkat cells cultured overnight with Ac₄GlcNAz with and without PUGNActreatment. PUGNAc is a commonly used inhibitor of O-GlcNAcase. SolubleJurkat lysate preparations were labeled with fluorescent alkyne. Lane 1)cells treated with 50 μM PUGNAc and 4 mM glucosamine 3 hrs prior toharvest; Lane 2) no treatment; Lanes 3-5) cells cultured overnight with250 μM Ac₄GlcNAz; Lanes 6-8) cells cultured overnight with 250 μMAc₄GlcNAz then treated with 50 μM PUGNAc and additional 250 μM Ac₄GlcNAz3 hrs prior to harvest. Proteins treated with PUGNAc (lanes 6-8) show amarked increase in O-GlcNAc staining over the untreated controls (lanes3-5). See FIG. 10.

Example 23 In-Gel Ligation of Glycoproteins

Fluorescent alkyne compounds for use in in-gel ligation are shown inFIG. 12 (A-D). Additionally, 2 potential fluorogenic alkynes are shownin E and F. The TAMRA-alkyne compound, shown in the upper left-handframe, was used in in-gel staining experiments whereby azido groups wereincorporated into proteins in vitro using a reactive azido-succinimidylester, or in vivo, by feeding cells azido-modified sugars.

Example 24 Azide-Alkyne Reaction Conditions for in-Gel or Western BlotDetection

The proteins should be in 50 mM Tris-HCl, pH 8.0 with 1.0% SDS.Final volume is 200 uL.

Component Volume Final Protein in 1% SDS, 50 mMTris pH8 50 uL 100-200 ugTris-HCl, pH 8.0 (1M) 7.5 uL 50 mM Propylene glycol 50 uL 25% CuSO₄ (50mM) 4 uL 1 mM DMSO, 0.5 M 4 uL 10 mM H20 62.5 uL to 200 uL Alkynecompound 2 uL 10 uM 1 mM (eg TAMRA or biotin) Na Ascorbate (100 mM) 10uL 5 mMCombine all components adding the ascorbate last. Vortex gently.Add 10 uL of 100 mM BCS (it will turn orange if CuI is present).Vortex gently.Layer with argon.Mix on rotator at room temperature for 1 hour.After the reaction, chloroform/methanol precipitate the protein usingthe following protocol:

200 uL Reaction 600 uL MeOH,. vortex 20 secs, (freeze 30′ if proteinamt. is low) 200 uL chloroform vortex 20 secs 450 uL H₂O vortex 20 secsSpin @ 18Kxg for 5 min Remove upper phase and add 450 MeOH. Vortex 20secs Spin @ 18Kxg for 5 min Remove supernatant and discard Add 600 uLMeOH only, vortex, and briefly sonicate to disperse pellet Spin @ 18Kxgfor 5 min

Na Ascorbate (mw 198) Dilution to 100 mM:

Na Ascorbate, dry, 5 mg H2O 250 uL

Cell lysates were obtained from cultured Jurkat cells that were fedazido-modified sugars. FIG. 13, shows the results of electrophoresis ofproteins labeled with the TAMRA-Alkyne compound using the protocolprovided. Lanes 2, 3, and 4 on the left side of the gel representcellular extracts that had incorporated azido-modified sugars, lane 1 isthe control, non-azide sugar fed cells. On the right, control azidelabeled proteins (ovalbumin and myoglobin) (+) or non-labeled controls(−) are shown at varying concentrations. The results show very efficientand selective in-gel detection of azido-modified proteins.

Example 25

2.5 μg each of azido-ovalbumin and azido-myoglobin were spiked into 80ug of unlabeled Jurkat lysate. The lysate was then labeled with TAMRAalkyne for 2 hrs. The reaction contained 50 mM TRIS pH8, 25% propyleneglycol, 1 mM CuSO₄, 5 mM sodium ascorbate, 20 uM TAMRA alkyne. Thereactions were performed with and without a chelator (10 mM of eitherTPEN, EDTA, bathocuproine disulfonic acid (BCS) or neocuproine). Thecontrol reaction was performed without CuSO₄. After labeling, thesamples were precipitated, resolubilized in 7 mM urea/2 mM thiourea/65mM DTT/2% CHAPS/and approximately 30 μg of each sample was analyzed on2-D gels (pH 4-7 IEF strips, 4-12% BIS-TRIS gels with MOPS buffer). TheTAMRA signal was imaged at 532 nm excitation, 580 long pass emission ona Fuji FLA3000 then the gels were post-stained with SYPRO® Ruby totalprotein gel stain (FIG. 14 A). The results show that addition ofchelator greatly improves the resolution of the protein separation.bathocuproine disulfonic acid (BCS), a Cu I chelator, gives the bestresults. See total protein stain, FIG. 14 B.

In a second experiment, the samples and click labeling conditions werethe same, except that chelator treatments included the addition ofeither 5 mM TPEN, BCS, or Neocuproine at the beginning of the reaction.After labeling, the samples were precipitated, resolubilized in LDSbuffer+5 mM TCEP and serial 2-fold dilutions were performed. Dilutionswere loaded onto 4-12% BIS-TRIS gels with MOPS running buffer (250 ngeach of ovalbumin and myoglobin in lane 1). FIG. 15 A shows that thechelators reduce the background of the image for the TAMRA signalwithout compromising sensitivity. In FIG. 15 B, post-staining withSypro® Ruby total protein gel stain shows that the band resolution ismuch better for the samples with chelator.

A further experiment testing the effect of chelators used the same clicklabeling conditions except that the chelator treatments includedaddition of either 7 mM, 5 mM, or 2 mM BCS; or 7 mM, 5 mM, or 2 mMneocuproine. The lanes marked with an asterisk in FIG. 16 indicatereactions in which the CuSO4 and BCS were added to the reaction andvortexed prior adding the sodium ascorbate. In all other reactions theCuSO4 and sodium ascorbate were added and vortexed prior to adding theBCS. The gels show that it is imperative to add the sodium ascorbate andCuSO4 to the reaction tube and mix prior to adding the chelator. If thechelator and CuSO4 are added and vortexed prior to adding the sodiumascorbate, the azide-alkyne labeling does not proceed, suggesting thatthe chelator inhibits the reduction of Cu (II) to Cu (I).

Example 26 Enzymatic Labeling of Antibodies Using Click Chemistry

Goat IgG antibodies were reduced and alkylated, then deglycosylated in 2separate aliquots using Endo Hf enzyme. Deglycosylated antibodies (2separate preps) were then labeled with GalNAz using 33 ng/uL Gal T1Y289L enzyme and 500 uM UDP GalNAz (0.5 ug/uL goat antibody) in a 150 uLreaction. Reactions were incubated at 4 degrees C. overnight. 4-500 ngof goat antibody (treated as listed on gel; either no-GalNAz control orazide labeled) was loaded into each lane of a 4-12% Bis Tris gel.Electrophoresis was performed at 200 v for ˜50 min. using MES buffer.Gels were stained with TAMRA-alkyne stain and imaged on the Fuji imagerat 532 nm (excitation) and 580 nm emission. Gels were poststained withSYPRO Ruby using the overnight protocol. See FIG. 17

Example 27 Synthesis of Cy™5.5Azide

To a solution of 6-(amino)-hexanyl-1-azide trifluoroacetic acid salt(see Scheme 1 for synthesis, 0.034 mmol) in DMF (0.1 mL) and DIEA (6.0L, 0.034 mmol) was added Cy™5.5 succinimidyl ester (5 mg, 3.4 nmol).After stifling the solution at RT for 10 min, the reaction solution wasconcentrated in vacuo. The crude was purified via HPLC.

Example 28 Synthesis of Cy™3Azide

To a solution of 6-(amino)-hexanyl-1-azide trifluoroacetic acid salt(see Scheme 1 for synthesis, 0.052 mmol) in DMF (0.1 mL) and DIEA (9.2L, 0.052 mmol) was added Cy™3 succinimidyl ester (5.0 mg, 5.2 nmol).After stirring the solution at RT for 10 min, the reaction solution wasconcentrated in vacuo. The crude was purified via HPLC.

Example 29 Synthesis of Cy™5.5Alkyne

To a solution of Cy™5.5 succinimidyl ester (GE Amersham, 5.0 mg, 3.7nmol) in DMF (0.1 mL) was added propargylamine (2.5 L, 0.037 mmol) andH₂O (0.2 mL). The solution was stirred at RT for 30 min thenconcentrated in vacuo. The crude was purified via HPLC.

Example 30 Synthesis of Cy™3Alkyne

To a solution of Cy™3 succinimidyl ester (GE Amersham, 5.0 mg, 5.7 nmol)in DMF (0.1 mL) was added propargylamine (3.9 L, 0.057 mmol) and H₂O(0.2 mL). The solution was stirred at RT for 30 min then concentrated invacuo. The crude was purified via HPLC.

Example 31 Metabolic Labeling with Azido Farnesyl Alcohol and Detectionwith Click-iT™ TAMRA Detection Reagent

COS7 and Jurkat cells were fed Lovastatin to suppress endogenousisoprenylation and azido-farnesyl alcohol (FN3-OH) for 36 or 72 hours.The dose was either 25 μM Lovastatin and 20 uM FN3-OH or 50 μMlovastatin and 50 μM FN3-OH. The media was replaced (including FN3-OH,but not lovastatin) for one set of samples cultured for 72 hours.Lysates were prepared by washing the cells 3 times with PBS and lysingwith 1% SDS/100 mM TRIS pH 8+protease inhibitors (1 mL of lysis bufferper 100 cm culture dish). The lysate was sonicated, heated at 70° C. for10 minutes then 200 μL aliquots were precipitated and frozen.Precipitated pellets were resolubilized with 50 μL of 1% SDS/100 mM TRISpH 8, labeled with the TAMRA Click-iT™ detection kit (C33370) and 20 μgwas analyzed on a 4-12% BIS-TRIS gel using MOPS buffer. The gels wereimaged on the BioRad FX imager using the 532 nm laser and 555 nm longpass emission filter. The gels were post-stained with SYPRO® Rubyprotein gel stain and imaged using the 488 nm laser and 555 nm long passemission filter.

Example 32 Metabolic Labeling with Azido Fatty Acid Analogs andDetection with “Click” Reagents

Azido-labeled fatty acid compounds, such as the palmitate compound shownbelow, or alkyne-labeled fatty acid compounds are added to culturedcells for a time period that allows the compound to enter the cells andbecome incorporated into fatty acid-modified macromolecules, includingproteins. Compounds that facilitate entry of the analogs into the cellscan also be used. After labeling, the cells are lysed and cellularazide- or alkyne-modified proteins are labeled using the appropriateclick partner probe in the presence of copper(I), or copper(II) in thepresence of a copper(II) reducing agent, a copper(I) chelating agentsuch as BCS, and an appropriate buffer to maintain optimal pHconditions.

Example 33 Metabolic Labeling with Azido Fatty Acid Analogs andDetection with “Click” Reagents

Azido-labeled fatty acid compounds, such as the palmitate compound shownbelow, or alkyne-labeled fatty acid compounds are added to cellularextracts in vitro, or under in vitro translation conditions. The fattyacylated proteins are detected by labeling the azide- or alkyne-modifiedproteins using the appropriate click partner probe in the presence ofcopper(I), or copper(II) in the presence of a copper(II) reducing agent,a copper(I) chelating agent such as BCS, and an appropriate buffer tomaintain optimal pH conditions.

Example 34 Palmitic Acid Azide Synthesis

The synthesis of palmitic acid azide is shown in the following reactionscheme.

To a solution of 16-bromohexadecanoic acid (0.50 g, 1.49 mmol) in DMSO(10 mL) was added sodium azide (0.14 g, 2.24 mmol). The reaction wasstirred at RT overnight. The solution was then cooled in an ice bath andquenched with water (10 mL). After stirring for 5 min, the ice bath wasremoved and the solution was allowed to warm to RT on its own. Theresulting solution was then extracted with diethyl ether (2×60 mL) andthe organic layers were combined, rinsed with saturated NaCl (3×10 mL),then dried over sodium sulfate. The solution was decanted, concentratedand purified by silica gel chromatography (10-25% EtOAc/hexanescontaining 1% acetic acid) to afford a clear, colorless oil (0.33 g,75%). TLC (25% EtOAc/hexanes) R_(f)=0.54, faint UV, brown spot iftreated with PPh₃/toluene then stained with ninhydrin. IR (KBr pellet)2126.9 cm⁻¹.

Example 35 Succinimidyl Ester Azide Synthesis

The synthesis of succinimidyl ester azide is shown in the followingreaction scheme.

6-(Boc-Amino)-hexanyl-1-p-toluenesulfonate

To a solution of 6-(Boc-amino)-1-hexanol (3.0 g, 13.8 mmol) in CHCl₃ (50mL) was added TEA (3.8 mL, 27.6 mmol) and p-toluenesulfonyl chloride(3.9 g, 20.7 mmol). The solution was stirred at RT overnight, dilutedwith CHCl₃ (200 mL), washed with H₂O (4×50 mL), rinsed with brine (1×50mL) and dried over Na₂SO₄. The solution was decanted, concentrated andpurified via silica gel chromatography (6.0×41 cm, 20-70% EtOAc/hexanes)to afford the product as a white solid (3.5 g, 69%). TLC (35%EtOAC/hexanes) R_(f)=0.72, UV active. 6-(Boc-Amino)-hexanyl-1-azide

To a solution of 6-(Boc-amino)-hexanyl-1-p-toluenesulfonate (3.2 g, 8.63mmol) in DMF (21 mL) was added sodium azide (1.12 g, 17.3 mmol). Thesolution was refluxed at 95° C. overnight. After cooling to RT, thesolution was diluted with Et₂O (160 mL) and washed with H₂O (100 mL).The aqueous layer was extracted a second time with Et₂O (100 mL) and thecombined organics were dried over Na₂SO₄. After decanting andconcentrating, the crude material was purified via silica gelchromatography (6×26 cm, 25-30% EtOAc/hexanes) to afford the product asa clear, colorless oil (2.0 g, 97%). TLC, (35% EtOAC/hexanes)R_(f)=0.74, brown spot with ninhydrin stain.

6-(Amino)-hexanyl-1-azide trifluoroacetic acid salt

To a solution of 6-(Boc-amino)-hexanyl-1-azide (0.2 g, 0.83 mmol) inCH₂Cl₂ (1.0 mL) was added TFA (1.0 mL). The solution was stirred at RTfor 2 h, evaporated to dryness and re-evaporated twice from toluene. Theproduct, 6-amino-hexanyl-1-azide trifluoroacetic acid salt (0.83 mmol)was used directly without further purification.

(N-6-Azido-hexanyl)glutaramide

6-Amino-hexanyl-1-azide (0.83 mmol) was dissolved in THF (1.0 mL) andN,N-diisopropylethylamine (0.29 mL, 1.65 mmol) was added. The solutionwas stirred at RT for 10 min then glutaric anhydride (0.47 g, 4.13 mmol)was added. The pale yellow solution was stirred at RT overnight. Thereaction solution was diluted with CHCl₃ (30 mL) and H₂O (10 mL), andacidified to a pH of 1 with 1% HCl; the organic layer was removed. Theaqueous layer was extracted two more times with CHCl₃ (2×30 mL). Thecombined organic layers were rinsed with brine (2×10 mL) and dried overNa₂SO₄. The solution was decanted, and concentrated. The crude waspurified via silica gel chromatography (10% MeOH/CHCl₃ containing 0.1%AcOH) to afford the product as a clear, colorless oil (0.16 g, 75%). Thecolumn was loaded with 10% MeOH/CHCl₃. TLC (10% MeOH/CHCl₃ with 0.1%AcOH) R_(f)=0.41, pink with p-anisaldehyde stain, no UV activity.

(N-6-Azido-hexanyl)glutaramide, succinimidyl ester

To a solution of the (N-6-azido-hexanyl) glutaramide (75 mg, 0.29 mmol)in THF (4.0 mL) was added pyridine (110 L, 1.36 mmol) followed bysuccinimidyl trifluoroacetate (200 mg, 0.95 mmol). The clear, colorlesssolution was stirred at RT for 4 h. The reaction solution was dilutedwith CHCl₃ (20 mL) and rinsed sequentially with 1% AcOH (2×5 mL), H₂O(2×5 mL) and brine (1×5 mL). The crude solution was dried over Na₂SO₄,decanted, and concentrated to afford the product as a clear, colorlessoil (0.10 g, 99%). TLC: (1:1, EtOAc/hexanes) R_(f)=0.64, orange withninhydrin, UV active.

Example 36 Succinimidyl Ester Alkyne Synthesis

The synthesis of succinimidyl ester alkyne is shown in the followingreaction scheme.

10-Undecynoic acid succinimidyl ester

To a solution of 10-undecynoic acid (0.40 g, 2.2 mmol) in CH₃CN (10 mL)was added O-(N-succinimidyl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate (0.99 g, 3.29 mmol). After stirring for 2 min at RT,the reaction was quenched with 1% AcOH and diluted with CHCl₃ (150 mL).The organic solution was then extracted with 1% AcOH (10 mL), rinsedwith H₂O (2×40 mL), then dried over Na₂SO₄. The solution was thendecanted and concentrated. A quantitative yield was assumed and thematerial was taken on directly to the next step. TLC (10% MeOH/CHCl₃)R_(f)=0.90, UV active.

tert-Butyl alkyne

To a solution of 10-undecynoic acid succinimidyl ester (0.61 g, 2.19mmol) in CH₃CN (8 mL) was added amino-dPEG™₂-tert-butyl ester (0.46 g,1.97 mmol, Quanta BioDesign) in CH₃CN (2 mL) at RT. After 2 hrs, thesolution was diluted with CHCl₃ (50 mL) and extracted with H₂O (5 mL).The aqueous layer was reextracted with CHCl₃ (2×50 mL). Combinedorganics were dried over Na₂SO₄, decanted and concentrated. The crudewas purified via silica gel chromatography (2.5% MeOH/CHCl₃) to affordthe product as a clear, pale yellow oil (0.48 g, 55%). TLC (9:1CH₃CN:H₂O) R_(f)=0.81.

Succinimidyl ester alkyne

To a solution of tert-butyl alkyne (0.48 g, 1.2 mmol) in CH₂Cl₂ (2.0 mL)was added TFA (2.0 mL). The solution was stirred for 1 h, thenconcentrated and reevaporated from toluene (2×1 mL). The resulting brownresidue was dissolved in CH₃CN (5.0 mL) and N,N-diisopropylethylamine(0.84 mL, 4.83 mmol) was added. The solution was stirred at RT for 2min, and then O-(N-succinimidyl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate (0.47 g, 1.56 mmol) was added. After 15 min thereaction was quenched and acidified with 1% AcOH to a pH of 4-5. Thesolution was extracted with CHCl₃ (3×50 mL). The combined organics werereextracted with H₂O (1×10 mL), then dried over Na₂SO₄, decanted andconcentrated to afford a tan solid (0.46 g, 87%). The crude material waspure enough for testing without further purification. TLC (8:2CH₃CN/H₂O) R_(f)=0.79.

Example 37 Iodoacetamide azide synthesis

The synthesis of Iodoacetamide azide is shown in the following reactionscheme.

6-(iodoacetamide)-aminohexanyl-1-azide

To a solution of 6-amino-hexanyl-1-azide trifluoroacetic acid salt (35mg, 0.14 mmol) in DMF (0.1 mL) was added iodoacetic anhydride (0.10 g,0.28 mmol) in the dark. After 2 hr, the reaction was stopped and thesolution was partitioned between CHCl₃ (10 mL) and H₂O (10 mL). Theorganic layer was removed and the aqueous layer was reextracted withCHCl₃ (1×10 mL). The combined organics were rinsed with saturated NaCl(1×5 mL), dried over Na₂SO₄, decanted and concentrated. Purification viasilica gel chromatography (2% MeOH/CHCl₃ containing 0.1% AcOH) providedthe product (35 mg, 81%) as a yellow oil. TLC (10% MeOH/CHCl₃)R_(f)=0.75.

Example 38 Iodoacetamide alkyne synthesis

The synthesis of Iodoacetamide alkyne is shown in the following reactionscheme.

N-(iodoacetamide)-propargylamine

To a solution of propargylamine in DMF was added iodoacetic anhydride inthe dark. After 2 hr, the reaction was stopped and the solution waspartitioned between CHCl₃ and H₂O. The organic layer was removed and theaqueous layer was reextracted with CHCl₃. The combined organics wererinsed with saturated NaCl, dried over Na₂SO₄, decanted andconcentrated.

Example 39 Maleimide alkyne synthesis

The synthesis of Maleimide alkyne is shown in the following reactionscheme.

Propargylamine Maleimide.

After the reaction of propargylamine and maleic anhydride in thepresence of TEA, the intermediate acid was cyclized in the presence ofacetic anhydride and sodium acetate at 70° C., to afford the desiredpropargylamine maleimide.

Example 40 Maleimide Azide Synthesis

The synthesis of Maleimide azide is shown in the following reactionscheme.

N-(6-Azido-aminohexyl)maleimide

After the reaction of 6-amino-hexanyl-1-azide trifluoroacetic acid saltand maleic anhydride in the presence of TEA, the intermediate acid wascyclized in the presence of acetic anhydride and sodium acetate at 70°C., to afford the desired N-(6-azido-aminohexyl)maleimide.

Example 41 Addition of Thioacetate-Azide and Amino-Azide FollowingBa(OH)2 Catalyzed β-Elimination of RII Phosphopeptide (NB B883-9-TN)

RII phosphopeptide (DLDVPIPGRFDRRVpSVAAE, 2192.08 Da) was azido-labeledby simultaneous β-elimination of the phosphate and addition of eitherthioacetate-azide or amino-azide to the resulting dehydroalanineresidue. 2 nmol of peptide and 3 μmol of azide tag was incubated in 30μL of 0.1M barium hydroxide for 2 hours at 35° C. After incubation, thebarium was precipitated with 7 μL of 0.5M ammonium sulfate. Aftercentrifugation, the supernatant was neutralized with 3 μL of glacialacetic acid, then desalted on a Vivapure RP microfilter. The peptide waseluted in 10 μL of 90% acetonitrile/0.1% TFA and analyzed by MALDI inpositive reflectron mode (spot 0.5 μL of eluate and 0.5 μL of 6 mg/mLα-cyano-4-hydroxycinnamic acid). Under these conditions, all of thepeptide is converted to addition product with thioacetate-azide as theaddition reagent, whereas conversion is incomplete with amino-azide asthe addition reagent. See FIGS. 18A and 18B.

TABLE 3 Thioacetate-azide

Amino-azide

Other catalysts that are not so basic but still effective in bringingabout the elimination include tertiary amines and hindered secondaryamines, such as but not limited to: phosphazene compounds,triethylamine, diisopropylethylamine, TEMED, triethanolamine2,2,6,6-teramethylpiperidine, Proton Sponge DABCO, N-methylmorpholine4-dimethylaminopyridine, 4-pyrrolidinylpyridine, an ion exchange resinsuch as Dowez 1 hydroxide, tetrabutylammonium hydroxide,benzyltriethylammoinum hydroxide, or other phase transfer bases calciumhydroxide, barium hydroxide, or gallium hydroxide (which will alsocomplex the phosphate group).

Example 42 Tagging Phosphoproteins Using Nucleotide Analogs

Phosphoproteins could be labeled in vivo or in vitro using alkyne orazide-tagged nucleotides whereby the azide or alkyne moiety is placed onthe gamma phosphate. For example one of the nucleotides shown below isadded to a reaction mixture containing a protein kinase and a kinasetarget molecule. After tagging the molecule is reacted with theappropriate alkyne or azide detection or affinity reagent forquantitation, visualization, or enrichment. In one example reaction,modified nucleotide substrates may be added directly to cultured cellsfor metabolic incorporation of the tagged gamma-phosphate molecule intocellular macromolecules including proteins. The process may involvetreatment of the cells with pharmacological agents to detect alterationsin phosphorylation dynamics. Entry of the compounds into live culturedcells could be enhanced by modifying the nucleotides with functionalgroups that would afford permeability, or by concomitant addition ofcell permeablizing agents. In another example reaction, the kinasereaction could be performed in vitro using cellular extracts as thesource of kinases and substrates. The modified nucleotides would beadded to the reaction mixture and the reaction mixtures incubated withor without the addition of pharmacological agents of interest. The invitro reaction may also entail adding an exogenous kinase or substratesource to the cellular extract along with the nucleotide analogs. Inanother application, the method could be used in vitro without cellularextracts, using purified kinases and kinase substrates. In all of thedescribed examples the reaction mix may contain a buffer optimized forthe particular kinases of interest, a kinase source, a metal ion source,glycerol, nucleotide ATP analog, and ATP. The “click” detection reactionwith an alkyne probe would be performed in the presence of copper(I), orcopper(II) in the presence of a copper(II) reducing agent, a copper(I)chelating agent, and an appropriate buffer to maintaining optimal pHconditions.

ATP-Alkyne:

ATP-Azide:

Example 43 Azido and Alkyne Reporter Molecules

5-TAMRA Azide.

To a solution of 6-(amino)-hexanyl-1-azide trifluoroacetic acid salt(see Scheme 1 for synthesis, 0.19 mmol) in DMF (0.5 mL) and DIEA (33 L,0.19 mmol) was added 5-carboxytetramethyl rhodamine, succinimidyl ester(5-TAMRA-SE, 50 mg, 0.094 mmol). After stifling the solution at RT for10 min, the reaction solution was concentrated in vacuo. The crude waspurified via silica gel chromatography (prep plate, 9:1 CH₃CN: H₂O) toafford the product as a pink solid (45.6 mg, 87%). TLC (CH₃CN: H₂O, 8:2)R_(f)=0.61, pink fluorescent spot; ESI-pos m/z 555 M⁺, C₃₁H₃₅N₆O₄(requires 555).

PEG-Biotin Azide.

To a solution of 6-(amino)-hexanyl-1-azide trifluoroacetic acid salt(see Scheme 1 for synthesis, 0.17 mmol) in DMF (0.5 mL) and DIEA (60 L,0.34 mmol) was added NHS-PEO₄-biotin (Pierce, 50 mg, 0.08 mmol). Afterstirring the solution at RT overnight, the solution was concentrated invacuo. The crude was purified via silica gel chromatography (7:1 CHCl₃:MeOH) to afford the product as a cloudy, white residue (12.3 mg, 12%).TLC (7:1, CHCl₃: MeOH, 8:2) R_(f)=0.54, faint UV active spot, stainspink with biotin dip; ESI-pos m/z 616 M⁺, C₂₇H₄₉N₇O₇S (requires 616).

Rhodamine Green™ Azide (Mix of 5- and 6-Isomers).

To a solution of 6-(amino)-hexanyl-1-azide trifluoroacetic acid salt(see Scheme 1 for synthesis, 0.20 mmol) in DMF (0.5 mL) and DIEA (50 L,0.28 mmol) was added Rhodamine Green™ carboxylic acid, succinimidylester, hydrochloride (mix of 5- and 6-isomers, 50 mg, 0.10 mmol). Afterstirring the solution at RT for 2 h the solution was concentrated invacuo. HPLC (Phenomenex Prodigy ODS, internal diameter 21.2 mm, eluent5-50% CH₃CN (over 60 min) in 25 mM TEAA, pH=4.7, flow rate of 20 mL/min)gave 21.1 mg of product (43%) t_(R)=43-47 min; TLC(CH₃CN: H₂O: AcOH,8:1:1) R_(f)=0.74, fluorescent yellow spot; ESI-pos m/z 499 (M+H,C₂₇H₂₇N₆O₄ requires 499).

Alexa Fluor® 488 Azide (5 Isomer).

To a solution of 6-(amino)-hexanyl-1-azide trifluoroacetic acid salt(see Scheme 1 for synthesis, 0.44 mmol) in DMF (0.5 mL) and DIEA (0.11mL, 0.88 mmol) was added Alexa Fluor® 4885-carboxylic acid,2,3,5,6-tetrafluorophenyl ester, bis(triethylammonium salt) (200 mg,0.22 mmol). After stifling the solution at RT for 1 h, the solution wasconcentrated in vacuo. HPLC (Phenomenex Prodigy ODS, internal diameter21.2 mm, eluent 0-60% CH₃CN (over 30 min) in 25 mM TEAA, pH=4.7, flowrate of 20 mL/min) gave 58.1 mg of product (30%) t_(R)=23-27 min; TLC(CH₃CN:H₂O, 8:2) R_(f)=0.58, fluorescent yellow spot; ESI-neg m/z 657(M⁻, C₂₇H₂₅N₆O₁₀S₂ ⁻ requires 657).

Alexa Fluor® 546 Azide.

To a solution of 6-(amino)-hexanyl-1-azide trifluoroacetic acid salt(see Scheme 1 for synthesis, 0.093 mmol) in DMF (0.5 mL) and DIEA (32 L,0.19 mmol) was added Alexa Fluor® 546 carboxylic acid, succinimidylester, (50 mg, 0.05 mmol). After stirring the solution at RT for 2 h,the solution was concentrated in vacuo.

HPLC (Phenomenex Prodigy ODS, internal diameter 21.2 mm, eluent 10-60%CH₃CN (over 60 min) in 25 mM TEAA, pH=4.7, flow rate of 20 mL/min) gave27.2 mg of product (54%) t_(R)=48-52 min; TLC (CH₃CN: H₂O, 9:1)R_(f)=0.24, fluorescent pink spot; ESI-neg m/z 1084 (M⁻,C₄₆H₅₅Cl₃N₇O₁₁S₃ ⁻ requires 1084).

Alexa Fluor® 594 Azide (5 Isomer).

To a solution of 6-(amino)-hexanyl-1-azide trifluoroacetic acid salt(see Scheme 1 for synthesis, 0.12 mmol) in DMF (0.5 mL) and DIEA (42 L,0.24 mmol) was added Alexa Fluor® 594 carboxylic acid, succinimidylester *5-isomer* (50 mg, 0.06 mmol). After stirring the solution at RTfor 2 h, the solution was concentrated in vacuo. HPLC (PhenomenexProdigy ODS, internal diameter 21.2 mm, eluent 25-60% CH₃CN (over 30min) in 25 mM TEAA, pH=4.7, flow rate of 20 mL/min) gave 16.5 mg ofproduct (32%) t_(R)=23-25 min; TLC (CH₃CN: H₂O, 9:1) R_(f)=0.36,fluorescent red spot; ESI-neg m/z 845 (M⁻, C₄₁H₄₅N₆O₁₀S₂ ⁻ requires845).

Dapoxyl Azide.

To a solution of 6-(amino)-hexanyl-1-azide trifluoroacetic acid salt(see Scheme 1 for synthesis, 0.25 mmol) in DMF (0.5 mL) and DIEA (43 L,0.25 mmol) was added Dapoxyl® carboxylic acid, succinimidyl ester (50mg, 0.12 mmol). After stirring the solution at RT for 1 h, the solutionwas concentrated in vacuo. Purified by SPE (Supelco C18 DSC) to give41.6 mg of product (78%); ESI-pos m/z 433 (M⁺, C₂₄H₂₈N₆O₂ requires 433).

Oregon Green® 488-Alkyne.

To a solution of Oregon Green® 488 carboxylic acid, succinimidyl ester(50 mg, 0.98 mmol) in DMF (0.5 mL) was added propargylamine (0.26 L,0.40 mmol) and H₂O (0.1 mL). After stifling at RT for 15 min, thesolution was concentrated. HPLC (Phenomenex Prodigy ODS, internaldiameter 21.2 mm, eluent

15-30% CH3Cn in 25 mM TEAA pH 4.7, flow rate of 15 mL/min) gave 44.5 mgof product (99%) t_(R)=5-13 min; TLC (CH₃CN: H₂O, 8:2) R_(f)=0.60,fluorescent yellow spot; ESI-neg m/z 448 (M−H⁺, C₂₄H₁₂F₂NO₆ ⁻ requires448).

Alkyne Biotin Compounds

Alkynyl-PEG-Biotin.

To a solution of NHS-PEO₄-biotin (Pierce, 25 mg, 0.004 mmol) in DMF (0.1mL) at RT was added propargylamine (0.3 mL, 4.5 mmol). After stiflingfor 3 h, the solution was concentrated in vacuo and re-evaporated twicefrom toluene. HPLC (Phenomenex Prodigy ODS, internal diameter 21.2 mm,eluent 35-50% MeOH in 25 mM NH₄Ac, pH 6.5, flow rate of 15 mL/min) gave14.4 mg, (64%, a white solid) t_(R)=26-30 min; TLC (CHCl₃:MeOH, 7:1)R_(f)=0.20, UV active spot; ESI m/z 529 (M+H⁺, C₂₄H₄₀N₄O₇S requires529).

Alkyne Dyes:

5-TAMRA-alkyne.

To a solution of 5-carboxytetramethyl rhodamine, succinimidyl ester(5-TAMRA-SE, 0.10 g, 0.19 mmol) in DMF (0.5 mL) was added propargylamine(25 L, 0.38 mmol) and H₂O (0.5 mL). After stirring the solution for 30min at RT, the solution was concentrated in vacuo. HPLC (PhenomenexProdigy ODS, internal diameter 21.2 mm, eluent 25-40% CH₃CN in 25 mMTEAA, pH 4.7, flow rate of 15 mL/min) gave 68 mg of product (82%, apurple solid) t_(R)=23-33 min; TLC (CH₃CN: H₂O, 8:2) R_(f)=0.67,fluorescent orange spot; ESI m/z 469 (M+H⁺, C₂₈H₂₆N₃O₄ requires 469).

Oregon Green® 488-Alkyne.

To a solution of Oregon Green® 488 carboxylic acid, succinimidyl ester(50 mg, 0.98 mmol) in DMF (0.5 mL) was added propargylamine (0.26 L,0.40 mmol) and H₂O (0.1 mL). After stifling at RT for 15 min, thesolution was concentrated. HPLC (Phenomenex Prodigy ODS, internaldiameter 21.2 mm, eluent 15-30% CH3Cn in 25 mM TEAA pH 4.7, flow rate of15 mL/min) gave 44.5 mg of product (99%) t_(R)=5-13 min; TLC (CH₃CN:H₂O, 8:2) R_(f)=0.60, fluorescent yellow spot; ESI-neg m/z 448 (M−H⁺,C₂₄H₁₂F₂NO₆ ⁻ requires 448).

Alexa Fluor® 532-Alkyne.

To a solution of Alexa Fluor® 532 carboxylic acid, succinimidyl ester(51 mg, 0.07 mmol) in DMF (4.0 mL) was added propargylamine (0.1 mL) andH₂O (1.0 mL). The solution was stirred at RT for 1 h then concentratedin vacuo to afford the crude product. HPLC (Phenomenex Prodigy ODS,internal diameter 21.2 mm, eluent 25-40% CH₃CN in 25 mM NH₄Ac, pH 4.7,flow rate of 15 mL/min) gave 30 mg of product (65%, a red solid)t_(R)=23-30 min; TLC (CH₃CN:H₂O, 1:1) R_(f)=0.58, fluorescent red spot;ESI m/z 664 (M⁺, C₃₃H₃₄N₃O₈S₂ requires 664).

Alexa Fluor® 488-Alkyne.

To a solution of Alexa Fluor® 488 carboxylic acid, succinimidyl ester,dilithium salt, mixed isomers, (51 mg, 0.08 mmol) in DMF (2.0 mL) wasadded propargylamine (54 L, 0.80 mmol). The solution was stirred at RTfor 4 h then concentrated in vacuo. The crude product was purified usingcolumn chromatography on silica gel (CH₃CN: H₂O, 8:2) to afford 20 mg(44%, an orange solid). TLC (CH₃CN: H₂O, 3:1) R_(f)=0.68; ESI-neg m/z570 (M−2, C₂₄H₁₆N₃O₁₀S₂ ²⁻ requires 570).

Alexa Fluor® 568-Azide.

To a solution of 6-(amino)-hexanyl-1-azide (see Scheme 1 for synthesis,0.04 mmol) in DMF (0.2 mL) and DIEA (7 L, 0.04 mmol) was added AlexaFluor® 568 carboxylic acid, succinimidyl ester (mix of isomers, 25 mg,0.02 mmol). After stifling the solution at RT for 2.5 h, H₂O (0.2 mL)was added and the solution was concentrated in vacuo. HPLC (PhenomenexProdigy ODS, internal diameter 21.2 mm, eluent 20-35% CH₃CN in 25 mMNH₄Ac, pH 4.7, flow rate of 15 mL/min) gave 15.3 mg of product (99%)t_(R)=24-30 min; TLC (CH₃CN: H₂O, 8:2) R_(f)=0.63, fluorescent pinkspot; ESI-neg m/z 817 (M−2, C₃₉H₄₁N₆O₁₀S₂ ⁻ requires 817).

Example 44 Triarylphosphine Dye

5-TAMRA-triarylphosphine

To a solution of N-Boc-triarylphosphine-amine (see Scheme 2 forsynthesis, 10 mg, 0.018 mmol) in CH₂Cl₂ (1.0 mL) was added TFA (0.5 mL).The reaction solution was stirred at RT for 30 min, concentrated invacuo, and re-evaporated twice from toluene. The crude amine (0.018mmol, 99%) was used directly in the next reaction without furtherpurification.

To a solution of triarylphosphine-amine (0.018 mmol) in DMF (0.2 mL) andDIEA (12 L, 0.089 mmol) was added 5-carboxytetramethyl rhodamine,succinimidyl ester (5-TAMRA-SE, 9 mg, 0.022 mmol). After stirring thesolution at RT for 2.5 h, the solution was concentrated in vacuo. HPLC(Phenomenex Luna C18(2), internal diameter 10 mm, eluent 40-55% CH₃CN in25 mM NH₄Ac, pH=7, flow rate of 5.0 mL/min) gave 4.1 mg of product (27%)t_(R)=32-34 min; TLC (MeOH:CHCl₃, 1:9) R_(f)=0.67, fluorescent pinkspot; ESI m/z 848 (M+H⁺, C₅₀H₄₈N₄O₇P requires 848).

The reagents employed in the examples are commercially available or canbe prepared using commercially available instrumentation, methods, orreagents known in the art. All references cited herein are incorporatedby reference in their entireties. The foregoing examples illustratevarious aspects of the invention and practice of the methods of theinvention. The examples are not intended to provide an exhaustivedescription of the many different embodiments of the invention. Thus,although the forgoing invention has been described in some detail by wayof illustration and example for purposes of clarity of understanding,those of ordinary skill in the art will realize readily that manychanges and modifications can be made thereto without departing from thespirit or scope of the appended claims.

U.S. patent applications with attorney docket numbers IVGN 745.1 andIVGN 745.2, both filed on Feb. 12, 2007, claiming priority to U.S.Provisional Application Nos. 60/772,221 and 60/804,640 are herebyincorporated by reference.

1-40. (canceled)
 41. A method of isolating a modified protein from aspent cell culture medium, the method comprising: incubating a cell in anutrient medium comprising an unnatural sugar which comprises a chemicalhandle; allowing the unnatural sugar to be added onto a protein byglycosylation, thereby forming the modified protein; allowing the cellto secrete the modified protein; harvesting the spent medium comprisingthe modified protein; and, isolating the modified secreted protein basedon the presence of the chemical handle.
 42. The method of claim 41wherein the chemical handle comprises an alkyne reactive moiety.
 43. Themethod of claim 42 wherein the alkyne reactive moiety is an azide. 44.The method of claim 43 wherein the azide comprises a GalNAz, ManNAz,GlcNAz, Ac₄GalNAz, Ac₄ManNAz, or a Ac₄GlcNAz.
 45. The method of claim41, wherein the cell comprises a eukaryotic cell, a mammalian cell, ahybridoma cell or a cell producing a recombinant protein.
 46. The methodof claim 45, wherein the mammalian cell comprises a murine cell, a plantcell, a goat cell, a rabbit cell, or a chinese hamster ovary (CHO) cell.47. The method of claim 41, wherein the modified protein is isolated onan activated solid or semi-solid support that comprises a group thatreacts with the chemical handle.
 48. The method of claim 47, wherein theactivated solid or semi-solid support comprises an alkyne, phosphine ora cyclooctyne group.
 49. The method of claim 47, wherein the solid orsemi-solid support comprises an agarose, an aerogel, a hydrogel, aresin, a bead, a biochip, a microfluidic chip, a silicon chip, amulti-well plate, a membrane, a conducting metal, a nonconducting metal,a glass, a magnetic support, a silica gel, a polymeric membrane, aparticle, a derivatized plastic film, a glass bead, cotton, a plasticbead, an alumina gel, a polysaccharide, a poly(acrylate), a polystyrene,a poly(acrylamide), a polyol, an agar, a cellulose, a dextran, a starch,a FICOLL, a heparin, a glycogen, an amylopectin, a mannan, an inulin, anitrocellulose, a diazocellulose, a polyvinylchloride, a polypropylene,a polyethylene, a nylon, a latex bead, a magnetic bead, a paramagneticbead, a superparamagnetic bead, or a starch.
 50. The method of claim 41further comprising: comparing a total number of modified proteins in thespent medium based on the presence of the chemical handle, with a totalnumber of secreted proteins in a second spent medium without theunnatural sugar, wherein the second spent medium is obtained from asecond cell cultured in a second nutrient medium in the absence of theunnatural sugar.
 51. A method for increasing the concentration of amodified protein comprised within a spent cell culture medium, themethod comprising: passing the spent cell culture medium over anactivated solid or semi-solid matrix to immobilize the modified proteinon the matrix; washing the immobilized modified protein under stringentconditions; treating the immobilized modified protein with a digestiveenzyme to release the immobilized modified protein from the matrix,wherein the activated solid or semi-solid matrix comprises a group thatreacts with the chemical handle on the modified protein.
 52. The methodof claim 51 wherein the modified protein comprises an unnatural sugarcomprising the chemical handle.
 53. The method of claim 52 wherein thechemical handle comprises an alkyne reactive moiety.
 54. The method ofclaim 53 wherein the alkyne reactive moiety is an azide.
 55. The methodof claim 54 wherein the azide comprises a GalNAz, ManNAz, GlcNAz,Ac₄GalNAz, Ac₄ManNAz, or Ac₄GlcNAz.
 56. The method of claim 51, whereinthe spent cell culture medium is obtained from culturing a cell thatcomprises a eukaryotic cell, a mammalian cell, a hybridoma cell or acell producing a recombinant protein.
 57. The method of claim 56,wherein the mammalian cell comprises a murine cell, a plant cell, a goatcell, a rabbit cell, or a chinese hamster ovary (CHO) cell.
 58. Themethod of claim 51, wherein the activated solid or semi-solid matrixcomprises an alkyne, phosphine or a cyclooctyne group.
 59. The method ofclaim 51, wherein the modified solid or semi-solid matrix comprises anagarose, an aerogel, a hydrogel, a resin, a bead, a biochip, amicrofluidic chip, a silicon chip, a multi-well plate, a membrane, aconducting metal, a nonconducting metal, a glass, a magnetic support, asilica gel, a polymeric membrane, a particle, a derivatized plasticfilm, a glass bead, cotton, a plastic bead, an alumina gel, apolysaccharide, a poly(acrylate), a polystyrene, a poly(acrylamide), apolyol, an agar, a cellulose, a dextran, a starch, a FICOLL, a heparin,a glycogen, an amylopectin, a mannan, an inulin, a nitrocellulose, adiazocellulose, a polyvinylchloride, a polypropylene, a polyethylene, anylon, a latex bead, a magnetic bead, a paramagnetic bead, asuperparamagnetic bead, a slide, an array, an agarose gel, apolyacrylamide gel, a polymeric particle or glass or a starch.